Abstract: A biomaterial silk fibroin is surface modified using antithrombin-Ⅲ（AT-Ⅲ） to improve its anticoagulant property. AT-Ⅲ was immobilized on the surface of silk fibroin/heparin blended membrane by NH₃ plasma treatment, followed by covalent cross-linking reaction. The immobilization efficiency was evaluated by protein surplus analysis method. The in vitro antithrombogenicity representing the activity of AT-Ⅲ was determined by the method of activated partial thromboplastin time（APTT）, prothrombin time （PT） and thrombin time （TT） test. The cytotoxicity of materials was tested by the MTT method. The results demonstrated that the AT-Ⅲ is immobilized on the membrane in an efficient and activity preservant form and, the anticoagulant performance of the modified fibroin membrane improved. These results anticipater the wider application of AT-Ⅲ, and provide new visions on the design of anticoagulant materials.