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.