Heart valve transplantation is a common procedure worldwide, often necessitated by valve calcification, particularly in older individuals. Mechanical and biological valves are the two main types used, each with its advantages and drawbacks. While mechanical valves offer durability, they require lifelong anticoagulant therapy due to clotting risks. Biological valves, while less durable, avoid the need for long-term medication but are prone to calcification. Glutaraldehyde (GA) treatment, commonly used to enhance biological valve properties, has limitations, including cytotoxicity and incomplete tissue penetration. This study aims to develop an alternative pre-treatment using Fetuin A, a serum protein known for its anti-calcification properties, particularly for elastin-rich tissues like bovine jugular veins. In vitro experiments using human vascular smooth muscle cells (VSMCs) cultured in calcification-inducing conditions confirmed Fetuin A’s ability to reduce calcium deposition by 20%. Additionally, GA-fixed bioprosthetic materials exhibited cytotoxicity, but this was mitigated by coating with amino acids like glycine, glutathione, or lysine, combined with sodium borohydride. Further enhancement of biocompatibility was achieved by coating with both amino acids and Fetuin A, showing promise for improving the performance of biological heart valve implants.