Cotton Fusarium wilt is one of the most serious disease in cotton production,which caused by Fusarium oxysporum f.sp.vasinfectum.Plant endophytes are important resources of biopesticides.Using tissue grinding and coating methods,the endophytes from Bulbophyllum sp. were separated.Antagonistic culture and agar column methods were used to screen the strains which could inhibit F. oxysporum f.sp.vasinfectum and was identified by molecular methods.The single-factor and Box-Behnken design were used to optimize fermentation process for producing antibacterial substances.The results showed that 13 strains were screened to have inhibitory effect,among which BBs-27 had the highest antifungal activity.The similarity between BBs-27 and Bacillus subtilis subsp.subtilis strain 168 was 98.93%.The optimal fermentation process were as follows:LB added to 2.5% sucrose,0.01% (NH₄)₂HPO₄ with pH=5,20 mL/50 mL of liquid volume,inoculation amount 2%,culture temperature of 40 ℃,rotation speed of 180 r/min,and fermentation time for 105 h.The verified inhibition rate on F. oxysporum f.sp.vasinfectum reached 88.06%.The half inhibition rate IC₅₀ of lipopeptide was 488.93 μg/L.Electron microscopic observation showed that lipopeptide produced by BBs-27 could make the hyphae of F. oxysporum f.sp.vasinfectum expand and irregular,and differentiate a large number of conidia.BBs-27 as a biocontrol bacterium has further research and development value.