Abstract: Fresh milk was selected as the raw material and Mucor miehei chymosin was used as the coagulant to prepare rennet casein.First,the effects of addition volume of enzyme and Ca Cl2,p H and temperature on the yield of rennet casein were studied using the single factor experiment. Subsequently,the technological parameters of casein production with milk- clotting enzyme from Mucor miehei were further optimized by Box- Behnken design.The results showed the optimum technological parameters were chymosin addition amount of 0.49%,Ca Cl2 addition amount of 0.37 mg / m L,p H6.08 and curd temperature of 35 ℃.Under the optimal conditions,the maximum yield of rennet casein was 3.39%. The study could provide reference basis for the full utilization of Mucor miehei chymosin in the production of rennet casein.