Abstract: Immobilized papain was prepared by alginate-modified gelatin. The effects of power,frequency,temperature and time of ultrasonic treatment to stimulate the activity of immobilized papain were investigated in this study. The results showed that the optimal ultrasonic conditions were:ultrasonic power was 0.35 W/cm²,ultrasonic frequency was 40 kHz,ultrasonic temperature was 60℃,and ultrasonic time was 40 min. Under the optimal conditions,the relative enzyme activity of immobilized papain was 273.23%. The results of validation experiments showed an actual value of 260.37%±12.23% with a relative error of 2.41% and high reliability. The activity of immobilized papain could be improved by reasonable ultrasonic treatment through this paper.