A preparation method of a gradient mineralized cancellous bone matrix material, comprising: processing naturally-derived bone tissue with an immunogenicity removal treatment for decellularization, and processing an obtained decellularized bone with an ultrasound gradient demineralization treatment to obtain the gradient mineralized cancellous bone matrix material and comprising specific steps of: Step 1: randomly selecting bone tissue from a mammal, removing the bone tissue with a drill, and cutting the bone tissue into cylindrical bone blocks with a scalpel; Step 2: then rinsing the bone blocks with sterile physiological saline for 2 hours before sterilizing by irradiation, wherein an irradiation dose is 5-40w; Step 3: rinsing the bone blocks with deionized water containing protease inhibitor to remove blood, fat tissue and other impurities, wherein a concentration of the protease inhibitor in the deionized water is 10-50KIU/ml, and the bone blocks are rinsed with the deionized water for 2-6 times and 3-20 minutes for each time; Step 4: separating the bone blocks into an embedding box, and putting the embedding box into a deionized water solution containing acetone and shaking for 1-4 hours, wherein a volume ratio of the acetone to deionized water in the deionized water solution is 10%-20%; Step 5: putting the embedding box into a deionized water solution containing tributyl phosphate and shaking for 1-4 hours, wherein a volume ratio of the tributyl phosphate to deionized water in the deionized water solution is 1%-5%; Step 6: putting the embedding box in a deionized water solution containing the protease inhibitor, shaking at 4°C for 24-48 hours with a shaker, and then freezing and thawing with liquid nitrogen for 2-6 cycles, wherein each cycle is from -80°C to 37°C, and a shaker speed is 50-300rpm; Step 7: putting the embedding box in a buffer solution containing Triton ® X-100, and shaking with a constant temperature shaker for 24 hours, wherein a concentration of the Triton ® X-100 is 0.5-5%; Step 8: shaking the embedding box in a buffer solution containing SDS with the constant temperature shaker for 36h, wherein a concentration of the SDS is 0.5- 10%; Step 9: putting the embedding box in a PBS buffer solution having a concentration of potassium chloride of 0.1-1M, and shaking with the shaker at 4°C and 100rpm for 2-12 hours; Step 10: shaking the embedding box in a PBS buffer containing potassium iodide with the shaker at 4°C and 100rpm for 2-12 hours, wherein a concentration of the potassium iodide in deionized water is 1-1.5M; Step 11: performing the ultrasound gradient demineralization treatment in a NaOH buffer solution containing EDTA2Na at 4°C-10°C for 4, 8, 12 and 24 hours, so as to obtain bone ECM (Extracellular matrix) materials with mineralization degrees of 100%, 90%, 60% and 0%; and Step 12: sterilizing the obtained materials by irradiation; wherein after each of the Steps 4-11, the deionized water is used to rinse for 6 hours before a next step. Step 1: randomly selecting bone tissue from a mammal, removing the bone tissue with a drill, and cutting the bone tissue into cylindrical bone blocks with a scalpel; Step 2: then rinsing the bone blocks with sterile physiological saline for 2 hours before sterilizing by irradiation, wherein an irradiation dose is 5-40w; Step 3: rinsing the bone blocks with deionized water containing protease inhibitor to remove blood, fat tissue and other impurities, wherein a concentration of the protease inhibitor in the deionized water is 10-50KIU/ml, and the bone blocks are rinsed with the deionized water for 2-6 times and 3-20 minutes for each time; Step 4: separating the bone blocks into an embedding box, and putting the embedding box into a deionized water solution containing acetone and shaking for 1-4 hours, wherein a volume ratio of the acetone to deionized water in the deionized water solution is 10%-20%; Step 5: putting the embedding box into a deionized water solution containing tributyl phosphate and shaking for 1-4 hours, wherein a volume ratio of the tributyl phosphate to deionized water in the deionized water solution is 1%-5%; Step 6: putting the embedding box in a deionized water solution containing the protease inhibitor, shaking at 4°C for 24-48 hours with a shaker, and then freezing and thawing with liquid nitrogen for 2-6 cycles, wherein each cycle is from -80°C to 37°C, and a shaker speed is 50-300rpm; Step 7: putting the embedding box in a buffer solution containing Triton ® X-100, and shaking with a constant temperature shaker for 24 hours, wherein a concentration of the Triton ® X-100 is 0.5-5%; Step 8: shaking the embedding box in a buffer solution containing SDS with the constant temperature shaker for 36h, wherein a concentration of the SDS is 0.5- 10%; Step 9: putting the embedding box in a PBS buffer solution having a concentration of potassium chloride of 0.1-1M, and shaking with the shaker at 4°C and 100rpm for 2-12 hours; Step 10: shaking the embedding box in a PBS buffer containing potassium iodide with the shaker at 4°C and 100rpm for 2-12 hours, wherein a concentration of the potassium iodide in deionized water is 1-1.5M; Step 11: performing the ultrasound gradient demineralization treatment in a NaOH buffer solution containing EDTA2Na at 4°C-10°C for 4, 8, 12 and 24 hours, so as to obtain bone ECM (Extracellular matrix) materials with mineralization degrees of 100%, 90%, 60% and 0%; and Step 12: sterilizing the obtained materials by irradiation; wherein after each of the Steps 4-11, the deionized water is used to rinse for 6 hours before a next step.