Abstract: In this study, single factor and response surface analysis were used to optimize the conditions for the production of Bacillus subtilis using Hongqu residue, the content of soluble sugar and soluble protein components during the fermentation were determined to analyze the limiting factors of bacterial growth, and the storage stability and the in vitro antioxidant activity of the microbioecologics were determined. The results showed that the highest yield of Bacillus subtilis in microbioecologics was (9.3±0.3) lg CFU/mL that was produced at 37 ℃ for 48 h with the conditions of 100 g/L Hongqu residue, and shaking speed of 220 r/min with 10% inoculation volume of 5 lg CFU/mL Bacillus subtilis. The content of soluble glucose in Hongqu residue was the main factor limiting the growth of Bacillus subtilis. When soluble sugar contents were low, the growth of Bacillus subtilis basically stopped. According to the accelerated preservation experiment, the viable count of probiotics stored at 20 ℃ for three months was 8.7 lg CFU/mL, which was higher than the criteria of microbioecologics application. The microbioecologics had strong antioxidant activity that scavenging capacity of DPPH free radical and hydroxyl free radical were 0.25 mg/mL (IC₅₀) and 0.69 mg/mL (IC₅₀) , respectively. This study proved that Hongqu residue could be used as a raw material to prepare Bacillus subtilis probiotics, which provided a practical basis for the sustainable green processing of microbial fermentation residue.