Abstract: The effects of alkaline,NPU,AX and flavourzyme on the peptide concentration,free amino acid content and degree hydrolysis of the hydrolysates from the WPI(whey protein isolate)were investigated. Based on the optimized conditions of alkaline,the addition time of NPU and flavourzyme were investigated and the optimal conditions of three-step-hydrolysis of composite enzyme were obtained. The optimal conditions of three-step-hydrolysis of composite enzyme were temperature 55℃,1.5% alkaline(E/S),pH8.5,and hydrolysis time 10 min. When pH was 7.2,1% NPU(E/S)was added. After WPI hydrolysis for 240 min with alkaline and NPU,1% flavourzyme(E/S)was added and hydrolysis continued for a further 20 min.The non-bitter WPI hydrolysates contained peptides(>60 g/L)and free amino acids(4348 mg/kg)with molecular weights below 1 kDa(69.52%).The IC₅₀ of ABTS and ACE were 1.02(0.377 μmol Trolox eq/mg)and 1.38 mg/mL,respectively. The WPI could be hydrolyzed by three-step-hydrolysis of composite enzyme to obtain the hydrolysates with antioxidant activity and ACE inhibition activity. This study laid a foundation for whey hydrolysates utilization in functional foods and health foods.