A　spherical　vinyl-functionalized　covalent-organic　framework　(COF-V)　was　prepared　at　room　temperature　by　a　facile　method　and　applied　as　a　novel　substrate　for　surface-enhanced　laser　desorption/ionization　mass　spectrometry　(SELDI-MS).　Compared　with　conventional　organic　matrices,　the　spherical　COF-V　with　high　crystallinity　and　good　monodispersity　exhibited　high　sensitivity,　no　matrix　background　interference,　wide-range　applicability,　high　salt　tolerance　and　reproducibility　in　the　characterization　of　small　molecules.　Considering　these　advantages,　the　applicability　of　the　spherical　COF-V-based　SELDI-MS　method　was　successfully　demonstrated　by　determining　trace　amounts　of　glucose　in　diabetic　urine,　which　would　be　a　promising　candidate　for　clinical　diagnosis　of　diabetes.　In　addition,　the　morphological　effect　and　the　desorption/ionization　mechanism　of　the　COF-V　were　investigated　in　detail　and　the　results　indicated　that　the　spherical　COF-V　substrate　could　greatly　enhance　the　LDI　process　compared　with　the　bulk　COF-V.　This　work　not　only　extends　the　application　of　COFs　in　MS,　but　also　offers　a　promising　alternative　for　small　molecule　identification　and　clinical　diagnosis　of　diabetes.　©　2020　The　Royal　Society　of　Chemistry.