Abstract: Trypsin, pepsin, papain, alcalase and neutrase were used for casein hydrolysis in this study. Pepsin was the best enzyme for the progress from the degree of hydrolysis ( DH) determination. In single factor experiment, hydrolysis temperature, p H, enzyme concentration and time was selected to detect the better conditions for pepsin hydrolysis with casein.Based on the results, by employing response surface methodology ( RSM) and building the mathematical model for hydrolysis conditions, the optimal hydrolysis conditions were obtained as: p H1.3, enzyme concentration 1.75 mg / m L and temperature 57℃ through the regression analysis and model evaluation. After the hydrolysis for 6h under the above conditions, the DH could reach 9.45%.