Abstract: [Objective]To optimize the spore formation conditions of an aflatoxin-degrading Bacillus subtilis strain N-1 a used in silage preparation by employing orthogonal experimental design.[Methods]The strain biomass and spore formation rate were used as the assessment index to evaluate the optimized condition combinations. The fermentation medium components and their corresponding ratio as well as the culture conditions were determined and optimized by using single factor experiment and orthogonal experimental design. [Results]The optimal ratio of the medium components were as followed： corn meal 2.0%,soybean cake powder 1.0%, MnSO4·H2O 0.10%, MgSO4·5 H2O 0.01%; the optimum spore formation conditions were as followed：p H value 7.0, fermentation duration 48 h, inoculation amount 4%, loading liquid volume 75 m L per 250 m L medium; under these conditions, the biomass of the N-1 a stain was 6.08 ×10^8 cfu/m L and the spore formation rate was 81.25%. [Conclusion]The determination of the optimal spore formation conditions of the N-1 a strain in shake flask production provided a theoretical basis for the pilot scale production.