Creatinine　(Cre),　a　metabolite　generated　by　muscles　and　kidneys,　holds　significant　importance　in　clinical　screening　and　detection　of　kidney　disease.　However,　the　existing　clinical　detection　of　Cre,　such　as　the　Jaffe　reaction-based　colorimetric　method,　requires　complex　sample　pretreatment　and　is　subject　to　interference　in　biological　samples.　Herein,　a　surface-assisted　laser　desorption/ionization　mass　spectrometry　(SALDI-MS)　method　based　on　a　donor-acceptor　covalent　organic　framework　(D-A　COF)　nanofilm　as　a　substrate　was　proposed　for　Cre　determination　in　human　serum.　The　D-A　COF　nanofilm　was　synthesized　using　a　solvothermal　reaction　on　indium-tin-oxide　(ITO)-coated　glass　plates,　which　featured　uniform　surfaces,　good　thermal　stability,　and　excellent　UV　absorption.　Compared　with　conventional　organic　matrices,　the　D-A　COF　nanofilm-based　LDI-MS　method　showed　low　background　interference　and　high　MS　response　and　was　successfully　used　for　the　analysis　of　low-weight　molecules,　such　as　amino　acids,　bisphenols,　and　estrogens.　On　this　basis,　the　D-A　COF　nanofilm-based　LDI-MS　method　was　developed　for　the　determination　of　Cre　in　human　serum.　This　method　showed　good　linearity　in　the　range　of　14.0-750.0　μmol　L−1　with　a　low　limit　of　detection　(LOD)　of　4.5　μmol　L−1,　making　it　suitable　for　the　determination　of　Cre　in　human　serum　with　different　concentration　levels.　This　work　demonstrates　the　potential　of　this　method　for　the　clinical　determination　of　Cre　in　human　serum　and　provides　a　new　direction　for　the　screening　and　determination　of　other　small-molecule　clinical　markers.　©　2025　The　Royal　Society　of　Chemistry.