Abstract: Immobilized papain was prepared through modified gelatin with glutaraldehyde.The effects of power,frequency,time and temperature on the efficacy of ultrasound treatment to stimulate the activity of immobilized papain were investigated in this study.The optimal ultrasound treatment conditions were determined by uniform design and least square regression analysis to be135 k Hz,0.05 W / cm2,20 min and 40 ℃.The results of validation experiments showed an actual value of 221.25% with a relative error of 3.58% compared to thearetical value. The optimal temperature and the optimal p H were not influenced by ultrasonic treatment.