Abstract: The single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and conditions of Bacillus subtilis K- 6- 9. The results indicated that the constituents components of medium, the filling volume of shaking flask, the rotation speed and the temperature were the major factors which affected the biomass yield of K- 6- 9. The sucrose 1.0%, peptone 0.25%, beef paste 0.5%, MgSO 4 0.2%, CaCl 2 0.1% and KH 2 PO 4 0.3% were selected to be the medium formula, and the fermentation conditions were the filling volume 50mL in 250mL shaking flask, inoculum 2%, 37℃, shaking at 180r /min for 18h, and initial pH at 7.0.In the shake flask test was carried out on the basis of 5L fermentor enlarge cultivation, 14h reached the growth peak, growth cycle 18h, the optimal put pot for 14h, at this time of spore was up to 1.40 × 109CFU/mL, which was 2.55 × 103times higher than that of initial fermentation condition