Establishing thrombosis-resistant surface is crucial to develop tissue-engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anti-coagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Cellular components of porcine carotid arteries were completely removed with chemical and physical means. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulphate salt and heparin-EDC. Bound heparin contents were measured by quantitative colorimetric assay of toluidine blue staining. The average content of heparin in treated vessels was 35.6 ± 11.6 mg/cm2 tissue, which represented 6.21 ± 2.03 UPS heparin/cm2 tissue. The stability of heparin linkage was tested by incubating the heparin-linked vessels either in PBS at 37°C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Anti-coagulation property of bound heparin was determined with a clotting time assay using fresh dog blood. Standardized small pieces of non-heparin-bound vessels were clotted in fresh dog blood within 10 min., whereas all heparin-bound vessels did not form clot during 1-hr observation. In vivo platelet deposition of the vessel was determined with a baboon model of the femoral arteriovenous external shunt and 111Indium labelling of platelets. There were 1.38 ± 0.07 × 109 and 0.64 ± 0.11×109 baboon platelets deposited on the control and heparin-linked vessels, respectively, at 60 min. These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue-engineered biological vascular grafts, which could be used for human coronary or low extremity artery bypasses.