To improve microbial activity of Bacillus subtilis NKY1145 fermentation broth, the medium composition（carbon, nitrogen and inorganic salts） were optimized in the experiment. The best combination of high density fer-mentation of Bacillus subtilis culture medium composition was ultimately determined, which was, 2% glucose, 3%molasses, 3% yeast powder, 2% soybean powder, 3% ammonium chloride, 2% magnesium sulfate and 3% sodium ci-trate. Maximum live bacteria number concentration of Bacillus subtilis NKY1145 could reach 6.3×10⁹CFU/m L.