Bacillus subtilis strain B47 is an endophytic bacterium of tomato and can produce substance to inhibit the growth of Bipolaris maydis which can cause southern corn leaf blight. The optimal nitrogen source, carbon source and salt for the production of antimicrobial substance by strain B47 were tested. The medium composition and fermentation conditions were optimized by orthogonal experiments. The results showed that the optimal nitrogen source, carbon source and salt were yeast extract, sucrose and MgSO4·7H2O, respectively. The best medium was YSB (Yeast extract-sucrose-beef extract). The composition of the medium was 2% (W/V) sucrose, 2% (W/V) yeast extract, 1.5% (W/V) beef extract, 0.06% (W/V) MgSO4?7H2O and 0.000 9% (W/V) FeSO4·7H2O. The optimal fermentation conditions were the combination of temperature 30 °C, initial pH 7.0, incubation time 6 d, 1% inoculum volume percentage and medium volume 40 mL/200 mL.