Strain B29 is Bacillus subtilis isolated from cucumber root perimeter soil, which is antagonistic to a variety of pathogenic fungi. Previous studies have found that fermentation culture of Bacillus subtilis can produce lipopeptide antibiotics with antibacterial activity. In order to optimize the fermentation process conditions to effectively promote the production and accumulation of lipopeptide antibiotics, the Box-Burman test was used to design three main factors affecting the lipopeptides: agitation rate, pH and temperature. The interactions between the main factors and the optimal conditions were determined by response surface analysis. The results showed that the theoretical yield of lipopeptide antibiotics could reach 763 μg·ml^-1 under the conditions of stirring at 348 r·min^-1, pH 6.9 and temperature 33 ℃, which effectively promoted the production and accumulation of antibacterial substances. The results provide a basis for further pilot scale up and industrial production, thus pave a new way for the biological control of plant diseases.