A new biomimetic material for artificial blood vessel with in situ catalytic generation of nitric oxide (NO) was prepared in this study. Organoselenium immobilized polyethyleneimine as NO donor catalyst and sodium alginate were alternately loaded onto the surface of electrospun polycaprolactone matrix via electrostatic layer-by-layer self-assembly. This material revealed significant NO generation when contacting NO donor S-nitrosoglutathione (GSNO). Adhesion and spreading of smooth muscle cells were inhibited on this material in the presence of GSNO, while proliferation of endothelial cells was promoted. In vitro platelet adhesion and arteriovenous shunt experiments demonstrated good antithrombotic properties of this material, with inhibited platelet activation and aggregation, and prevention of acute thrombosis. This study may provide a new method of improving cellular function and antithrombotic property of vascular grafts. nitric oxide, catalytic generation, electrospinning, vascular graft, biocompatibility Citation:An J, Chen SY, Gao JC, Zhang X, Wang YY, Li YD, Mikhalovsky S, Kong DL, Wang SF. Construction and evaluation of nitric oxide generating vascular graft material loaded with organoselenium catalyst via layer-by-layer self-assembly. Sci China Life Sci, 2015, 58: 765 -772,