Aim To produce specific monoclonal antibody (McAb) against human vascular endothelial cell membrane proteins for investigating the functions and relation to disease of vascular endothelial cells. Methods McAb was prepared with classical hybridoma technique. Human vascular endothelial cell line(ECV304)was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third days after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with cellular enzyme-linked immunoabsorbent assay (CELISA). Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using fast protein liquid chromatography(FPLC),and its Ig class, subclass and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Results One line of hybridoma cells with high expression of specific McAb termed as NH-2 against vascular endothelial cell membrane protein was obtained. The Ig subclass of the McAb NH-2 was IgG1 and the titer of ascitic McAb was 1×10-6. Furthermore, the content of ascitic McAb was 18.82 g/L. Flow cytometry, CELISA and western blot assays demonstrated that McAb NH-2 could specifically recognize antigen expressed on ECV304 and human umbilical vein endothelial cell(HUVEC). Meanwhile, the tissue specificity and cell specificity of antigen recognized by McAb NH-2 were identified better seprately by immunohistochemical ABC staining and flow cytometry. On the other hand, the molecular weight of antigen protein recognized by McAb NH-2 was about 40kD. Conclusions NH-2 can specifically recognize the natural antigen expressed mainly in vascular endothelial cells, which will potentially be usefull for investigation of the functions and clinic application values of vascular endothelial cells.