<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with Wdesk from Workiva-->
<!--Copyright 2022 Workiva-->
<!--r:ac7b3184-40e4-480a-848a-62cafec14019,g:0193ddaa-4dfc-4929-a51b-5cabdd5c6d44-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://www.nautilus.bio/role/Cover" xlink:type="simple" xlink:href="naut-20220503.xsd#Cover"/>
  <link:definitionLink xlink:role="http://www.nautilus.bio/role/Cover" xlink:type="extended" id="i6a44182528d9445bae8a3823987e81af_Cover">
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressAddressLine1_0889fa8c-60ef-4e1f-a8ab-b61d368fc50e" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressAddressLine1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:to="loc_dei_EntityAddressAddressLine1_0889fa8c-60ef-4e1f-a8ab-b61d368fc50e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressCityOrTown_de520f48-03c3-4fae-9cff-65ba099a8e42" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressCityOrTown"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:to="loc_dei_EntityAddressCityOrTown_de520f48-03c3-4fae-9cff-65ba099a8e42" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressStateOrProvince_4f9bbc79-c248-4bb2-8a35-7ba3968c700e" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressStateOrProvince"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:to="loc_dei_EntityAddressStateOrProvince_4f9bbc79-c248-4bb2-8a35-7ba3968c700e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressPostalZipCode_6ce1da54-2e99-4f1e-b916-767278a6bb37" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressPostalZipCode"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:to="loc_dei_EntityAddressPostalZipCode_6ce1da54-2e99-4f1e-b916-767278a6bb37" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_DocumentPeriodEndDate_5cbf1794-6cc1-4a81-abe3-8a32178f2b2e" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_DocumentPeriodEndDate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:to="loc_dei_DocumentPeriodEndDate_5cbf1794-6cc1-4a81-abe3-8a32178f2b2e" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressesTable_277a575b-baf2-4a4b-a9d6-3ea5a6449391" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_dei_EntityAddressesLineItems_154df5b6-8106-4d9c-a003-800e46ab4849" xlink:to="loc_dei_EntityAddressesTable_277a575b-baf2-4a4b-a9d6-3ea5a6449391" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_EntityAddressesAddressTypeAxis_8994eb49-4417-4921-a015-256abd54908b" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_EntityAddressesAddressTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_dei_EntityAddressesTable_277a575b-baf2-4a4b-a9d6-3ea5a6449391" xlink:to="loc_dei_EntityAddressesAddressTypeAxis_8994eb49-4417-4921-a015-256abd54908b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_AddressTypeDomain_8994eb49-4417-4921-a015-256abd54908b_default" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_AddressTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_dei_EntityAddressesAddressTypeAxis_8994eb49-4417-4921-a015-256abd54908b" xlink:to="loc_dei_AddressTypeDomain_8994eb49-4417-4921-a015-256abd54908b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_AddressTypeDomain_d87ab774-6372-4cc7-976f-34d17f1665c4" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_AddressTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_dei_EntityAddressesAddressTypeAxis_8994eb49-4417-4921-a015-256abd54908b" xlink:to="loc_dei_AddressTypeDomain_d87ab774-6372-4cc7-976f-34d17f1665c4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_dei_FormerAddressMember_16e2cf4e-730d-4f8d-ba3e-94b67a8f9c99" xlink:href="https://xbrl.sec.gov/dei/2021/dei-2021.xsd#dei_FormerAddressMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_dei_AddressTypeDomain_d87ab774-6372-4cc7-976f-34d17f1665c4" xlink:to="loc_dei_FormerAddressMember_16e2cf4e-730d-4f8d-ba3e-94b67a8f9c99" xlink:type="arc" order="0"/>
  </link:definitionLink>
</link:linkbase>
