Abstract: In order to screen out Bacillus sp. with high production of antimicrobial lipopeptide and determine its optimal fermentation conditions, the study isolated and identified antimicrobial lipopeptide-producing Bacillus sp. strains from traditional fermented soybean paste, and adopted orthogonal test to investigate how lipopeptide production by Bacillus sp was affected by fermentation conditions, such as fermentation inoculum amount, fermentation filling volume, fermentation time and fermentation temperature. The results showed that 27 strains of Bacillus sp. were isolated and screened out from 9 portions traditional fermented soybean paste of the Northeast, among which 6 strains were identified by 16S rDNA as having the genes sfp, fenB and ituA for synthesizing lipopeptide. According to the determination of lipopeptide yield and inhibition effect, Bacillus subtilis SN-20 and Bacillus amyloliquefaciens SN-46 showed excellent performance with the yield of 106 and 72 mg of lipopeptide per unit, and strong inhibition effect on both gram-positive and negative indicator bacteria. The optimal fermentation process of Bacillus subtilis SN-20 was found to be 3% inoculum, 20% fermentation filler, 36 h fermentation time and 32 ℃. The optimal fermentation process of Bacillus subtilis SN-46 was 2% inoculum, 40% fermentation filler, 24 h fermentation time and 32 ℃. Under these conditions, the unit biomass yields of lipopeptides for the two strains of Bacillus before optimization were 106.11 and 76.23 mg/g, respectively. After optimization, they increased by 21.85% and 23.84%, respectively. The study results effectively increased the production of antimicrobial lipopeptides from Bacillus.