Abstract: This paper applied the combination of superfine grinding technology and ultrasonic technology to the research of the effect of the protein hydrolysis of whey pepsin.The impact factors including superfine grinding orders, ultrasonic power, ultrasonic time were investigated by response surface design based on the single factor experiment, with the content of soluble protein as the responses, also the mathematical models were established. The results showed that the best experiment combination factors of hydrolysis effect were superfine grinding orders 2000 of order, ultrasonic power of 250 W, ultrasonic time of 50 min.The significance of every experiment factors on the content of soluble protein was decreased according to ultrasonic power > ultrasonic time > superfine grinding orders. Under the optimal experiment combination factors, the content of soluble protein was 6.03 mg/m L, which was 1.39 times of the content of soluble protein that was not dealt with experiment combination factors.