Abstract: [Objective] To increase sporulation rate and spore concentration and offer the theoretical foundation for industrial production of Bacillus megaterium preparation. [Method] Fermentation culture medium and culture conditions of Bacillus megaterium JD-2 were studied by single factor test and orthogonal test. [Result] The optimum medium formula was composed of 10.0 g/L corn starch, 5.0 g/L peptone, 5.0 g/L yeast extract, 5.0 g/L NaCl, 1.0 g/L CaCO3 and 0.5 g/L MgSO4·7H2O. The optimum culture conditions are as follows： initial spore concentration, 10^6 CFU/mL; initial pH value, 7.0; culture temperature, 30 °C; shaking speed, 200 r/min; and volume, 25 mL medium in a 250 mL flask. When enlarge fermentation was performed in a 15 L auto fermentor with dissolved oxygen level less than 30%, cell concentration reached 3.50×10^9 CFU/mL, spore concentration reached 3.40×10^9 CFU/mL and sporulation rate reached 97.2% after 22 h culture. [Conclusion] The optimal culture conditions can be applied in practical production.