In this study， the pre-treated whey protein by ultrasound was used as the substrate for enzymatic hydrolysis. OPA and ELISA method were used to analyze the optimal conditions of ultrasonic pretreatment - enzymatic hydrolysis for whey protein by the crude enzyme of Kluyveromyces marxianus Z17， in order to reduce the antigenicity of whey protein (characterized by the antigenicity of α-LA and β-LG). The results showed that the hydrolysis degree of whey protein was significantly affected by initial pH value and hydrolysis temperature， and the antigenicity of α-LA and β-LG was apparently influenced by initial pH value， and the interaction of ultrasonic interval time and ultrasonic power also played a role in affecting the antigenicity of α-LA and β-LG. The optimal conditions for enzymatic hydrolysis to transform whey protein by Kluyveromyces marxianus Z17 were obtained by response surface method: the reduction rates of α-LA antigenicity and β-LG antigenicity were predicted to reach the maximum at the ultrasonic interval time of 16 s， the ultrasonic power of 400 W， the initial pH of 6.16 and the enzymatic hydrolysis temperature of 18.48 ℃， which were 65.56% and 57.96%， respectively.