Abstract: To improve the spore yeild of Bacillus mucilaginous in submerged fermentation, optimizing process of the culture conditions including single factor experiments and orthogonal experiment was carried out and spore yeild according to blood cell counts board was used as an indicator. The results showed that the optimal culture conditions were:inoculation amount of 5%, initial pH of 7.5, CaCO3 amount of 6g/1000mL, broth volume of 70mL/250mL flask, rotate speed of 250r/min, temperature of 32℃ for the beginning 24h, then increased to 37℃ for 12h further, and incubation time of 36h totally. Under the optimal culture conditions, the spore yeild of Bacillus mucilaginous was 9.3×109cfu/mL at 36h using blood cell counts board.