Based on the chicken by-products as raw materials， the preparation of peptone was optimized by the single factor combined with the quadratic general rotary unitized design. With the peptone yield as the index， using trypsin and alkaline protease to hydrolysis chicken by-products， and the enzyme solution was optimized by using quadratic general rotary unitized design. Established four yuan quadratic regression model with analysis of DPS software， and the optimal factors for the double enzyme ratio was 1.3 ∶ 1 （mass ratio）， enzyme solution temperature was 54 ℃， enzyme solution pH was 8.2， enzymolysis time was 3.4 h. Under the condition， the actual peptone rate values was 29.28%.