Abstract: To study enzymatic properties of chymosin from mihei pusillus, the optimal temperature, pH and thermal stability of chymosin were investigated. Along with this, the effects of metal ions and enzyme amount on the activity of the enzyme were examined, and the proteolytic activity, Km and Vm of the enzyme were measured. The optimal reaction temperature and pH of the enzyme were 70℃ and 6. Incubation at 65℃ for 10min resulted in 96% loss of enzyme activity. Ferrous ion was an enzyme activator which could improve the enzyme activity. In contract, Copper ion was an inhibitor of the enzyme, Sodium ion and Potassium ion had less effect on the enzyme activity. The measurement results would be lager by reducing the added amount of the enzyme. The proteolytic activity, relative, Km and Vm of the enzyme were 1048U/g, 190. 84g/L, 7. 8678g/s.