Abstract: In recent years, Bacillus used in aquaculture has attracted much attention as a microbial candidate to maintain the health and well-being of aquatic animals. Bacillus licheniformis FA6 is a probiotic isolated from the intestines of grass carp (Ctenopharyngodon idella), and has been shown improve growth performance, promote immune response and help the host digest through its enzyme activity. However, the fermentation process of B. licheniformis FA6 is unclear, which has limited its large-scale application in aquaculture. In this study, both traditional one-factor-at-a-time method and response surface methodology were used to optimize the fermentation medium and conditions based on the viable count of B. licheniformis FA6. We found that the optimal fermentation medium of B. licheniformis FA6 contained 70 g/L soluble starch, 40 g/L casein peptone, 2 g/L K₂HPO₄, 1 g/L KH₂PO₄, 2 g/L Na₂HPO₄, 2 g/L MgSO₄ and 8 g/L NaCl. The optimal fermentation conditions were an initial pH of 7.0, inoculation quantity of 1%, fermentation temperature of 28.8℃, fermentation time of 31h, liquid volume of 50 mL/250 mL and rotational speed of 160 r/min. Under these optimized conditions, the viable count of B. licheniformis FA6 reached 7.97×10⁹ CFU/mL, which was 8.1 times higher than before optimization. The acquisition of the optimal fermentation process of B. licheniformis FA6 provides a reference for the high-density production and industrial application of fish-derived Bacillus.