Tissue engineering might bear promising solutions for overcoming the limitations of biological and mechanical heart valve substitutes. The concept of heart valve tissue engineering bases on decellularized biological matrices, as removal of cellular components might reduce immunological reactions, which are thought to be responsible for accelerated valvular graft deterioration and their subsequent repopulation with autologous cells, which leads to tissue integrity and continuous remodeling. Here, we report a method for efficient removal of cells from ovine heart valve tissue (including cusp, wall, and myocardial cuff) by detergents for the generation of biological valvular scaffolds.