In order to improve sporulation rate of Bacillus coagulans, in this study, we investigate the high-density fermentation culture conditions for Bacillus coagulans using a batch experiments including single-factor experiment, Plackett-Burman design experiment, steepest ascent search experiment, response surface experiments with the Box-Behnken design. The results showed that Bran 36.91 g/L, corn steep powder 13.70 g/L, MnSO4 0.84 g/L were the optimal medium for Bacillus coagulans. Under this condition, the spore number of QR-N323 was 2.848×1010 cfu/mL. The number of spores increased by 61.72% compared that before the optimization. The optimized medium components were used for 500 L fermentation scaling-up test with temperature 40 ℃, Rotating speed 150 r/min, ventilatory capacity 2.5 m3/h, inoculation quantity 5%, pH 7.0 and incubation time 36 h. The results were comparable to the optimized experiment results, which provides necessary data supportting further production process optimization.