Abstract: To prepare the milk-clotting enzyme with high efficiency,stepwise salting-out combined with aqueous two-phase extraction was used to purify the enzyme from Dregea sinensis Hemsl. The effects of the best purification ratio of polyethylene glycol(PEG)/salt and the amount of enzyme on the purification of crude enzyme were investigated. The purity and molecular weight of enzyme was determined by SDS-PAGE. The results showed that when the saturation range of ammonium sulfate was 20%~40%,the specific activity of crude enzyme was 167.846 MCA/mg. When the relative molecular weight of PEG was 6000,the mass fraction of ammonium sulfate was 20.57%,the mass fraction of ammonium sulfate was 14.74%,and the amount of enzyme was 1.0 mL,the activity of the enzyme was increased by 1.87-fold. The purity of the enzyme measured by SDS-PAGE was higher and the molecular weight was about 25 kDa. High-purity enzyme can be obtained by separation and purification of the aqueous two-phase,which can provide reference value for the subsequent large-scale production and application of the enzyme from Dregea sinensis Hemsl.