Immobilization of heparin on biomaterials surface has been proven to be an effective strategy for preventing thrombus formation. However, the procedures of most immobilization methods (physical adsorption, covalent linkage, electrostatic interaction) are complicated and time-consuming. In the present study, heparin with various concentrations immobilized on a titanium (Ti) substrate via polydopamine layer for improving blood compatibility was investigated. Water contact angle measurement showed that the immobilization of heparin resulted in an increase of the hydrophilicity. X-ray photoelectron spectroscopy (XPS) and Toluidine Blue O (TBO) tests displayed that the heparin molecules were successfully immobilized on Ti surface. The evaluations of blood compatibility (hemolysis rate, APTT, platelet adhesion and activation, fibrinogen conformational change) showed that the immobilization of heparin decreased hemolysis rate, prolonged blood coagulation time, reduced platelets adhesion and activation, and induced less fibrinogen conformational change. Moreover, a significant inhibition of blood coagulation and platelet adhesion was obtained when the heparin concentration was higher than 5 mg/mL, indicating that only with a certain surface densities could heparin perform its anticoagulant properties well. The results suggest that the immobilization of heparin via polydopamine layer can confer excellent antithrombotic properties, and the heparin immobilization method via polydopamine layer provides an alternative approach for other biomolecules immobilization on biomaterials surface. Thus it is envisaged that this method will be potentially useful for the surface modification of blood-contacting biomaterials.