Abstract: Chymosin is the key enzyme in making cheese. In order to produce highly active recombinant bovine chymosin ( B-chy) , the linearized recombinant pGAPZαA-B-pchy was transformed into Pichia pastoris GS115 by electro-transformation.The transformed strain, in YPD culture media with glucose as a carbon source, could secret bovine prochymosin.SDS- PAGE analysis showed that the expressed bovine chymosin had a molecular mass of37 ku which was the expected size. After 96 h of fermentation, the chymosin activity reached 96 SU /mL. Enzymatic properties analysis showed that the optimal temperature for milk- clotting was 60℃. The chymosin was relatively stable below 50℃ and in pH2~6.When the Ca2 +concentration was 40 mmol /L, calcium could promote the enzyme reaction to the highest level, then with the increase in Ca2 +concentration, the enzyme activity gradually decreased.Metal ions, such as Al3 +, Mn2 +, Fe2 +, Mg2 +and K+, could significantly increased the chymosin activity.However, Co2 +, Zn2 +, Cu2 +and Ni+significantly inhibited the chymosin activity. This study optimized the conditions for chymosin expression, which provided a theoretical foundation for the industrial production of chymosin.