Multilayer films consisting of polyethylenimine (PEI) and heparin were successfully prepared on biomedical 316L stainless steel surface via electrostatic self-assembly (ESA) of the PEI and heparin. The process of ESA of PEI/heparin was monitored by static contact angle, electrochemical impedance spectroscopy (EIS), reflection adsorption spectroscopy and X-ray photoelectron spectroscopy data. The contact angle and EIS data revealed that the multilayer coating was stable in Tris-HCl (pH 7.35) buffer solution for 21 days. The static platelet adhesion and static clotting time experiments indicated that the PEI/heparin-deposited stainless steel could resist the platelet adhesion and prolong the static clotting time effectively. Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices.