The endothelialization of the metal surface of vascular stents came into focus as a new method for improving the biocompatibility of intravascular stents. This article has its focus on building a biofunctional layer on the activated titanium surface with anti-CD34 antibody, vascular endothelial growth factor (VEGF), and heparin by a layer-by-layer (LBL) self-assembly technique, to promote the endothelialization of the surface. When compared with titanium surface, the number of adhered endothelial progenitor cells (EPCs) on LBLs increased 47.1% after 5 days culture, meanwhile the proliferation rate of EPCs on LBLs during 5 days culture also exhibit significantly improvement. The results of blood compatibility evaluation clearly show that the LBLs reduce the number of adhered and activated blood platelets (adhered: Ti 83% vs. LBL <20% and activated: Ti 57% vs. LBL 19%), and the activated partial thromboplastin time of the LBL surface prolonged about 20 s in compared with platelet-poor plasma. The assembled LBL thus improves the thromboresistance of the titanium surface. The presented biofunctional multilayer of anti-CD34, VEGF, and heparin on titanium can significantly improve the blood compatibility and the endothelialization of a medical device. The biofunctional layer supports generation of a new endothelium onto titanium surface by capturing EPCs and oriented differentiation.