Abstract: In order to reduce the cost of antibacterial peptide production in fermentation industry and to improve the yield of antibacterial peptide Bacillomycin D,Plackett-Burman design was utilized to evaluate the significant nutritional factors which affect the yield of Bacillomycin D in the original medium 6010 on the basis of the single factor experiment. And the industrial culture medium was optimized by the Central Composite Design response surface method. The results showed that the most three significant factors were maltose syrup,urea and MgSO₄·7H₂O. The optimum production was obtained when the cells grown in media containing:Maltose syrup 46 g/L,urea 0.72 g/L,MgSO₄·7H₂O 0.35 g/L,corn steep liquor 16 mL/L,(NH₄)₂SO₄ 3 g/L,K₂HPO₄ 7 g/L,MnSO₄·H₂O 0.05 g/L. After optimization,the yield of Bacillomycin D fermented by Bacillus subtilis M364 reached(620.2±13.9) mg/L,which increased by 50% compared to the non-optimized medium(415.5±9.8) mg/L and 2.6 times compared to the Landy fermentation medium(240±20.6) mg/L. The yield of Bacillomycin D in fermenter was(890.6±20.1) mg/L,which increased by 44% compared with the optimized production under the shaking flask condition and 1.14 times compared to the original 6010 medium. The culture medium provided by this study could achieve high yield,cost down and shorten fermentation time.