Abstract: Taking defatted and decalcified bone powder as raw material, the extraction of gelatin was studied by ultrasonic-assisted enzymatic hydrolysis using pepsin. The effects of ultrasonic treatment defatted and decalcified bone powder before, during and after enzymatic hydrolysis ( different ultrasonic time and ultrasonic power) on the yield of gelatin were compared.The single factor experiment results showed that the yield of gelatin could be significantly increased by ultrasonic treatment before enzymatic hydrolysis and during enzymatic hydrolysis.The yields of gelatin reached 81.35% and 83.32% respectively at 250 W for 20 min and 250 W for 10 min by ultrasonic treatment before enzymatic hydrolysis and during enzymatic hydrolysis respectively, which was higher than without ultrasonic treatment, 9.96% and 11.93% respectively.However, the yield of gelatin was only 71.59% by ultrasonic treatment at 300 W for 10 min after enzymatic hydrolysis, which had no advantage compared with without ultrasonic treatment. SEM showed that the surface of defatted and decalcified bone powder appeared small holes when treated with ultrasonic before enzymatic hydrolysis and during enzymatic hydrolysis.The surface of defatted and decalcified bone powder became smoother when treated with ultrasonic after enzymatic hydrolysis. It was indicated that the structure of defatted and decalcified bone powder with ultrasonic treatment before enzymatic hydrolysis and during enzymatic hydrolysis could be loosed. SDS-PAGE showed that ultrasonic-assisted enzymatic hydrolysis had no effect on the molecular weight distribution of gelatin. Ultrasonic-assisted enzymatic hydrolysis, which improved the yield of gelatin, could provide useful reference in industrial production of bone gelatin.