Abstract: Bacillus subtilis CS16 could effectively inhibit banana fusarium wilt pathogen.The formation conditions for CS 16 were optimized in this study.By single factor orthogonal experiment,the fermentation medium and culture conditions were selected to improve the antibacterial activity of CS 16.The results showed that the optimal culture conditions were fermentation medium consisting of corn flour 0.5%,soybean meal 1.5%,K2HPO40.1%,with culture pH 7.5,inoculation valum 4%,culture fluid volume 50mL/250 mL.In which,by culturing 24hat30℃,the biomass of CS 16 reached to 3.3 × 109 cfu· mL-1 that was 42 times higher than that of control.Meanwhile,the antibacterial activity of CS 16 increased by 7%.It was found that antimicrobial compounds of CS 16 could be grouped into two types:to kill the pathogenic cells or to inhibit the growth of pathogenic hyphae.