Abstract: The fermentation media components and culture conditions of the thermostable protease from a thermophilic strain Bacillus subtilis BY25 were further studied. A six factor and three level orthogonal experiment was conducted to observe the effects of fermentation medium components on thermostable protease yield. Wheat bran was replaced by soybean cake powder for its low contribution rate. Single factor experiment was carried out to optimize the protease production. The optimized culture condition was at 30 ℃, with initiating pH of 8.0, 4.5%-5% inoculum size for 72 h; and the optimized fermentation medium components were described as follows:glucose 60 g/L, soybean cake powder 60 g/L, Na₂HPO₄ 4 g/L and CaCl₂ 0.5 g/L. After fermentation, the casein hydrolysis activity of the enzyme was estimated up to 101.1 μmol/(min·mL), much higher than other domestic reports.