Although　matrix-assisted　laser　desorption/ionization　mass　spectrometry　(MALDI-MS)　has　become　a　ubiquitous　and　effective　tool　for　macromolecules,　direct　analysis　of　small　molecules　by　MALDI-MS　using　conventional　organic　matrices　poses　a　challenge.　Herein,　a　large-area,　uniform,　and　stable　covalent　organic　framework　(COF)　nanofilm　prepared　directly　on　indium-tin　oxide　(ITO)　glass　was　first　introduced　as　a　substrate　for　LDI-MS,　which　showed　enhanced　sensitivity,　no　background　interference,　and　high　reproducibility　in　the　analysis　of　diverse　small　molecules.　Taking　into　account　all　these　merits,　an　attractive　approach　of　COF　nanofilm-based　LDI-MS　was　developed　to　quantitatively　evaluate　the　pharmacokinetics　of　5-fluorouracil　(5-FU)　in　mouse　plasma.　A　good　linear　relationship　(10-20,000　ng/mL)　and　a　low　limit　of　detection　(LOD)　for　5-FU　(∼100　pg/mL)　were　achieved.　In　view　of　the　fact　that　the　COF　nanofilm　was　uniform　and　without　the　requirement　of　additional　matrix　spraying,　it　was　further　extended　for　LDI-MS　imaging　(LDI-MSI)　to　visualize　the　spatial　distribution　of　5-FU　in　mouse　liver　at　different　interval　times　after　intravenous　and　intragastric　administrations.　The　results　indicated　that　the　decay　of　5-FU　in　mouse　liver　obtained　with　the　COF　nanofilm-based　LDI-MSI　was　consistent　with　the　tendency　of　5-FU　pharmacokinetics.　This　work　not　only　offers　an　alternative　solution　for　LDI-MS/MSI　analysis　of　small　molecules　but　also　extends　the　application　fields　of　COF　nanofilm　in　MS　research.　©　2021　American　Chemical　Society.