Objective: This study aimed to explore the optimal medium composition and culture conditions for obtaining the maximum viable count of Brevibacillus laterosporus. Methods: Based on establishing the correlation regression equation between the MTT colorimetric method and plate counting method, the optimal medium composition (carbon source, nitrogen source, inorganic salt) and fermentation conditions (initial pH, temperature, inoculation amount, KH₂PO₄) for obtaining the maximum viable count of B. laterosporus were optimized. Results: A significant linear correlation for viable count determination between MTT colorimetry and plate counting (R²>0.999) was found; Maltose, CaCl₂, initial pH and KH₂PO₄ were the significant factors. The optimal fermentation condition was 8.75 g/L maltose, 0.17 g/L CaCl₂, 7.07 initial pH and 3.73 g/L KH₂PO₄. Under the control of these conditions, the number of viable counts was 8.12×10⁸ CFU/mL. However, no significant difference from the theoretical number (8.25×10⁸ CFU/mL) was found. Conclusion: Based on MTT colorimetry and response surface methodology, the culture conditions for the maximum viable count of B. laterosporus were optimized and the number of viable counts was higher than before optimization increased by 3.02 times.