Bacillus subtilis Tpb55 was isolated from tobacco phyllosphere and had antagonistic activity against to tobacco brown spot. Cultivation medium of Tpb55 was researched by single factor and orthogonal experiments. The fermentation conditions tested included fermentation medium, pH value before sterilized, fermentation temperature and fermentation time. The results showed that the optimal cultural liquid consisted of 1% glucose (10 g), 0.3% peptone (3 g), 0.05% Mgso4 (0.5 g), 0.15% yeast extract (1.5 g) and 0.15% (1.5 g) beef extract. The optimal fermentation conditions of stain Tpb55 were predicted as below: temperature 31℃, pH7.0 and 72h, agitation 180r/ min.