A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.