Abstract: Bacillus subtilis L7 that was isolated from cow dung and the antibacterial substance in its metabolites had strong inhibition on multi-pathogenic bacteria. The studies on the fermentation conditions for producing the antibacterial substance showed that the optimal fermentation conditions were as follows：pH of 7.0, culturing with 4% inoculum amount under constant-temperature oscillation incubator for 60 h at 30 ℃. The antibacterial substance in its metabolites was stable at 80~ 121 ℃ for 30 min under pH of 6.0~10.0. The antibacterial activity decreased under illumination conditions after 2~6 h. In conclusion, the fermented products of strain L7 had stronger stability, so it could be preserved under the environment of lucifuge,neutral pH and room temperature for a short term.And it might be used as a new kind of natural antiseptic in food industry or applied in the production of anti-infection micro -ecological preparation after treatments of increasing the activity and purifying.