Construction of heparinylated multilayer films on Ti-O via streptavidin/biotin interaction was conducted in the present study. An organic layer of 3-aminopropylphosphonic acid (APP) was first introduced on Ti-O by self-assembling, and then biotin was immobilized by photochemical methods. So streptavidin and biotinylated heparin were assembling through biorecognition, and a desired 6-layer heparinylated multilayer was obtained through layer-by-layer driven by streptavidin/biotin interaction. The in vitro platelet adhesion and activation were investigated by a static platelet adhesion test. The clotting time was examined by activated partial thromboplastin time (APTT). Results show that the heparinylated multilayer coated Ti-O can significantly decrease platelet adhesion and activation, and prolong clotting time of APTT compared to untreated Ti-O, which indicates the heparinylated multilayer coated Ti-O displays more excellent anticoagulation performance than that of the bare Ti-O. (C) 2012 Published by Elsevier B.V.