Abstract: Bacillus subtilis HS-09 has good probiotic performance and has been used as a microbial agent in aquaculture industry. In order to improve the spore-producing fermentation level of the strain HS-09, with the spore yield as the index, the carbon and nitrogen sources of the fermentation medium of spore produced by Bacillus subtilis HS-09 were screened and optimized by single factor tests and orthogonal experiments to determine the best fermentation medium. The results showed that the primary and secondary factors affecting the spore yield of Bacillus subtilis HS-09 were in order as follows:corn flour, ammonium sulfate and glucose. The optimized fermentation medium of spore produced by Bacillus subtilis HS-09 was as follows:glucose 10 g·L^-1, ammonium sulfate 8 g·L^-1, corn flour 15 g·L^-1, magnesium sulfate 0.5 g·L^-1, manganese sulfate 0.5 g·L^-1, dipotassium phosphate 1 g·L^-1, monopotassium phosphate 2 g·L^-1, and calcium carbonate 3 g·L^-1. The optimal spore-producing fermentation medium was used for the amplified fermentation in 200 L fermentation tank, and the effective clump count of fermentation could reach 4.3×10⁹ cfu·mL^-1, achieving a high conversion rate of 90% of spore. The results of this study could provide reference for the industrial production of the strains.