Abstract: The condition for liquid fermentation of an Aspergillus niger strain was optimized by using Design-Expert software combined with the Box-Behnken Design (BBD) and Response Surface Methodology (RSM) analysis method, and the Ralstonia solanacearum was used as indicator and inhibition zone Aspergillus niger aganst was used as the response value. The optimum carbon and nitrogen for Aspergillus niger were obtained through single factor experiment, which were maltose and wheat bran, respectively. Through Plackett -Burrman design, some factors of fermentation medium were evaluated and three factors had prominent effect on inhibition zone, which were MgSO4, CaCl2 and Tween 80, respectively. Then steepest ascent was used to approach maximum inhibition zone diameter. The BBD and RSM were used to determine the optimal levels of the main influencing factors, as the composition of fermentation medium was optimized as follow: maltose 25.00 g/L, MgSO4 0.38 g/L, K2HPO4 0.10 g/L, KCl 0.50 g/L, CaCl2 0.05 g/L, FeSO4 0.20 g/L, wheat brain 8.50 g/L,yeast paste 1.50 g/L, Tween 80 0.46 mL/L. Under those optimal conditions, the diameter of inhibitory zone increased by 51.23% from 24.79(依0.72) to 37.49(依0.34) mm.