Background and Objectives Glutaraldehyde (GA) has been used as a representative method of tissue preservation in cardiovascular surgery. However, GA has showed limited durability including calcification, mechanical failure and toxicity. To overcome those unsolved problems, we analyzed the crosslinking differences of primary amines, GA and genipin in their mechanical and biochemical properties with a single or double crosslinking agent for clinical application. Materials and Methods Samples were divided into 3 groups; control, single crosslinking fixation and double crosslinking fixation after decellurarization using bovine pericardium. For analysis of the biochemical and mechanical properties of each crosslinking method, tensile strength, percentage strain, thermal stability, resistance to pronase, nynhydrin and cytotoxicity test were studied. Results Combined hexamethylene diamine and suberic acid in the carbodiimide hydrochloride/N-hydroxysucinimide solution (EDC/NHS) after decellurarization, tensile strength and strain percentage were not statistically significant compared to the single crosslinking treated groups (p>0.05). Tissue crosslinking stability was weak in single treatment of diphenylphosphoryl azide, suberic acid, low concentration of EDC, hexamethylene diamine and procyanidin groups, but thermal stability and resistance to the pronase and ninhydrin were markedly increased in concentrated EDC/NHS or after combined double treatment with low concentration of GA or genipin (p<0.001). Conclusion Single or double crosslinking with low concentration of carbodiimide, diphenylphosphonyl azide, procyanidin, suberic acid and hexane diamine were not as effective in mechanical, biochemical, cytotoxic and crosslinking properties compared to GA or genipin fixation, but their mechanical and chemical properties were much improved when combined with low concentrations of GA or genipin in the double crosslinking process.