Abstract: To optimize the fermentation conditions for a feed probiotics, Brevibacillus brevis YSJ-0401, which was found to inhibit Escherichia coli obviously. The best fermentation conditions for highest inhibition of YSJ-0401 were studied based on time, temperature, shaking speed and medium pH. The results showed that its inhibition zones against E. coli were 10.00-13.00 mm after cultured for 24-84 h, 9.00-14.00 mm at 24-39 ℃, 13.75-15.00 mm under the shaking speeds of 90-210 r/min and 10.50-14.50 mm at pH values of 6.0-8.0, respectively. In conclusion, the best fermentation conditions for YSJ-0401 against E. coli were as follows: pH 7.5, 30 ℃, 48 h culture time and 90-210 r/min shaking speed.