The invention discloses a preparation method of a tendon conjunction bone decellularization material of a natural tissue source. The heel tendon conjunction bone tissue of any size of a mammal is treated by normal saline containing protease inhibitor, PBS containing protease inhibitor, a PBS buffer solution containing Triton X, a PBS buffer solution containing SDS and a PBS buffer solution containing DNA enzyme, and the tendon conjunction bone decellularization material is obtained. Complete decellularization treatment can be carried out on tendon cells and bone cells on tendon conjunction bone tissue and cartilage cells on the interface at the same time, conditions are mold, no ECM damage is caused, the process is rapid and stable, and the obtained tendon conjunction bone material has the advantages of being good in biocompatibility, strong in plasticity, high in biomechanical strength and the like, and can be used for repairing tendon degenerative diseases caused by various etiological factors clinically, tendon severe tear and tendon conjunction bone regeneration and repair obstacles.