In order to optimize fermentation conditions for production preparation of Yak blood antioxidant peptides with Bacillus subtilis, ·OH scavenging rate was taken as index to analyze the effects of fermentation time, inoculum concentration, substrate concentration on the antioxidant result with response surface methodology. The optimum fermentation conditions were determined as followsfollowed: 75 g/L of substrate concentration, 69.5 h of fermentation time, 2.5%(V/V) of inoculum concentration. Under such conditions, the ·OH scavenging rate was 74.48%, which was close to the theoretical value of 75.77%. Eventually, the content of peptides in fermented supernatant was 2.31 mg/mL.