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The nutritional and environmental conditions on antibacterial substance production of Bacillus amyloliquefaciens BI2 were investigated. Through single factor experiments,the optimum carbon and nitrogen sources were obtained. Three important factors were selected by Plackett Burman,. Path of steepest ascent was undertaken to approach the optimal region of the significant factors. Box-Behnken design and response surface analysis were adopted to further investigate mutual interaction between variables. The results showed that the optimum conditions were as follows: Soluble starch 0.5%, Corn syrup 1.2%, NaNO3 1.2%, Sodium glutamate 1.58%, Tween 80 0.104%, medium volume 71.05mL. Under these conditions, the inhibition ratio reached 98.3%.