The Arg-Gly-Asp (RGD) sequence is one of the most effective cellular adhesion ligands and has been used to improve cellular activity of biomaterials for tissue regenerative medicine. Aim of this study was to develop new facile immobilization methods of RGD on electrospun polyurethane (PU) meshes using tyrosinase (Ty) to promote attachment and proliferation of human umbilical vein endothelial cells (HUVECs). The PRGDGGGGGY (PRGD-Y) peptides were readily immobilized on PU substrates for 1 h using 0.4 KU/mL of Ty. Water contact angles and X-ray photoelectron spectroscopy (XPS) was used to verify that the PRGD-Y peptide was effectively conjugated on the PU (0.120 nmol/mgmesh) and the surface concentration was shown to be proportional to the peptide feed concentration. Enhanced HUVEC attachment on the polymer substrate was observed after 1 day and the immobilized PRGD-Y was clearly demonstrated to promote proliferation and spreading of the cells on the surface, indicating that the method developed for the conjugation of the peptides on the PU was very efficient. These results suggest that this enzyme-triggered immobilization method for the cellular adhesion ligand, RGD, onto the PU mashes may be an efficient tool to improve the biological activity of substrates especially for vascular tissue engineering.