Abstract: Three psychrotrophic strains coded Px-1, Px-3 and Px-5 which could produce protease and have obvious hydrolysis circles on defatted milk powder plate, that were isolated from raw milk. The stains were isolated again by time-enzyme activity curve. The strain Px-1 had the strongest protease activity. Composition of fermentation medium was optimized by quadratic design. The optimum fermentation medium composition was as following:4% sucrose, 4% alpha lactose, 2% glucose, 0.7% yeast extract powder, 0.5% casein, 0.3% tryptone and 0.05% tween 80. Under the optimum conditions, the protease activity of Px-1 could reach 42.5451U/mL, higher than the value before optimization 30.2291U/mL.