Chitosan as an antibacterial agent and heparin as an anti-adhesive agent were alternatively deposited onto aminolyzed poly (ethylene terephthalate) (PET) films to construct anti-adhesive and antibacterial multilayer films. The contact-angle and UV data verified the progressive buildup of the multilayer film by alternate deposition of the polyelectrolytes. The properties of multilayer films were investigated by contact angle, atomic force microscopy (AFM), lateral force microscopy (LFM) and UV spectra. The results of initial adhesion of Escherichia coli (E. coli) on PET substrates showed that the number of E. coli adhered onto the control PET was in a much greater extent than onto the chitosan/heparin multilayer films, and the number of adhesive bacteria decreased with a decrease in assembly pH. The in vitro antibacterial test indicated that a multilayer of chitosan/heparin could kill the bacteria effectively. The number of viable bacteria decreased by 7% after 7h in contact with the control PET films, but by 46-68% for the multilayer-modified PET films. Only 3-8% of the cells were viable for the multilayer-modified PET films after 24 h. It is interesting to find the assembly pH has a remarkable effect on the antibacterial property of the multilayer. The number of viable bacteria on the multilayer assembled at pH1/4 3.8, 2.9 and 6.0 decreased by 68%, 58% and 46%, respectively. Such an easy processing and shape-independent method to prepare an anti-adhesive and antibacterial surface may have good potential for surface modification of cardiovascular devices.