Deoxynivalenol(DON), as a secondary metabolite of some fusarium species, has been harmful to the food and feed industry. To improve spore-producing ability of Bacillus subtilis, the fermentation pH, temperature, supplemental carbon source and duration of Bacillus subtilis that efficiently degraded DON were studied in 5L-quadruple fermenters. The best fermentation conditions were determined, the fermentation pH was controlled at 7.5 and the temperature was controlled by stages, kept at 37 ℃ for first 12 hours, then raised to 39 ℃ until the end; 10 g/L molasses was added once at the 12th hour, then the fermentation was terminated at the 30th hour. Under the optimal conditions, the number of spores could reach 2.9×1010 CFU/mL and the spores’ rate could reach 96.7%. The fermentation liquid were further prepared into direct inoculated microbial inoculum and applied to remove DON from grain processing by-products; Under the condition that dry matter content in the composite material culture medium was not more than 30%, and the inoculation amount of the bacterium preparation was more than 0.1%, DON in the grain by-products could be effectively removed by fermentation and the highest degradation rate could reach 98.8%.