The invention discloses a preparation method of a natural-tissue-derived decellularized and decalcified bone material. According to the method, any bone tissue of a mammal is treated with a protease inhibitor-containing normal saline buffer, an organic solvent, Tirton X-containing PBS (phosphate buffer saline), SDS (sodium dodecyl sulfonate)-containing PBS, pancreatin-containing PBS, deoxyribonuclease-containing PBS, an EDTA (ethylene diamine tetraacetic acid) isotonic solution and ultrasonic waves, and the decellularized and decalcified bone material is obtained. Cancellous bones and cortical bones can be decellularized completely and simultaneously, the conditions are mild, damage to an ECM (extracellular matrix) is avoided, rapidness and stability are realized, and the obtained decellularized and decalcified bone material has the advantages of good biocompatibility, high plasticity, high biomechanical strength and the like and can be used for clinically repairing bone regeneration and repair disorder such as bone defects, nonunion and the like caused by various causes.