RationaleHomocysteine　(hcy)　is　a　metabolite　in　the　human　body　and　an　important　determinant　of　cardiovascular　health.　To　assist　in　the　assessment　of　human　cardiovascular　safety,　a　rapid　and　accurate　quantitative　analysis　method　must　be　developed.　Laser　desorption/ionization　mass　spectrometry　(LDI-MS)　has　been　widely　used　in　biological,　chemical,　and　medical　analyses　due　to　its　excellent　characteristics　of　high　throughput,　low　sample　consumption,　and　high　speed.　Here,　an　LDI-MS　method　based　on　covalent　organic　framework　(COF)　film　was　developed　for　the　rapid　and　sensitive　determination　of　hcy　in　human　serum　using　an　isotope-labeled　internal　standard.　MethodsWe　tried　to　cultivate　COFs　on　indium-tin　oxide　substrate　at　room　temperature　to　form　a　thin　film,　which　was　used　in　LDI-MS.　Compared　with　the　traditional　organic　matrices,　the　COF　film　had　a　clean　background　and　a　high　signal　response　for　the　detection　of　hcy.　In　addition,　using　COF　film　as　the　substrate,　with　a　high　signal　intensity　and　a　clean　background,　can　be　helpful　to　analyze　a　series　of　small　molecules　such　as　amino　acids,　bisphenols　(Bps),　estrogens,　and　drugs.　We　evaluated　the　limit　of　detection　(LOD)　and　the　reproducibility　of　this　method.　Finally,　the　calibration　curve　was　constructed　for　the　quantification　of　serum　hcy　using　an　isotope-labeled　internal　standard　and　was　applied　for　the　rapid　determination　of　hcy　in　clinical　human　serum.　ResultsCOF　film-assisted　LDI-MS　had　a　higher　response　signal　and　a　cleaner　background　compared　to　four　conventional　organic　matrices.　The　LOD　of　the　method　for　hcy　was　0.5　mu　mol/L,　equivalent　to　500　fmol.　This　method　exhibited　excellent　performance　for　small-molecule　compounds,　including　amino　acids,　Bps,　drugs,　and　estrogens.　The　reproducibilities　of　this　method　for　shot-to-shot　and　dot-to-dot　were　determined　to　be　good.　This　method　was　applied　for　the　rapid　analysis　of　hcy　in　clinical　human　serum,　showing　good　correlation　with　those　obtained　by　hospital　testing.　ConclusionThe　COF　film-based　LDI-MS　method　had　simple　sample　preparation,　short　analysis　time,　clean　background,　and　good　reproducibility　for　hcy　analysis.　As　an　important　indicator　of　human　health,　the　detection　of　serum　hcy　is　of　great　significance　to　human　health.