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The fermentation medium of brevibacillin by Brevibacillus laterosporus was optimized. Six main factors which affected brevibacillin production were evaluated by Placket-burman design. Three significant factors, sucrose, beef extract and Mg2+, were selected and the optimal area of the response surface was determined by the steepest climbing experimental; the optimal medium components were obtained by using a three-factor and three-level Box-Behnken experiment. The optimal medium components for B. laterosporus producing brevibacillin were: Sucrose 28.65 g/L, beef extract 17.04 g/L, Mg2+13.01 g/L, tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L. Under this optimum condition the yield of brevibacillin was 457.87±5.12 µg/mL, which was 34.6% higher that before optimization, and it was very close to the maximum value 462.94 µg/mL predicted by the theory.