Abstract: The production of water- soluble protein powder was studied by mixed fermentation of Bacillus subtilis N-2 with milt and soybean meal as culture medium.Five factors, including fermentation time, inoculum volume, solid- water ratio, fermentation temperature and initial p H were studied by single- factor experiments, in order to analyze the effect on water- soluble substance dissolution rate, water- soluble protein extraction rate, acid- soluble protein extraction rate, free amino acids content and nucleic acid content of protein powder. Fermentation conditions were optimized by response surface methodology based on the single- factor experiments.The optimum fermentation condition was as follows: fermentation time of 60h; solid- water ratio of 1 ∶ 1.4; initial p H of 7.2, fermentation temperature of 36℃, inoculum volume of 8%. In this fermentation process, water- soluble substance dissolution rate was 80.25%, water- soluble protein extraction rate was 91.86%, acid- soluble protein extraction rate was 57.25%, free amino acids content was 25.9%, and nucleic acid content was 11.2%.The proportion of essential amino acids in total amino acids increased significantly in the protein powder which was prepared under the optimum conditions.