The medium compositions of Bacillus species (three strains) were optimized sequentially through two statistical experimental designs, Plackett-Burman and the Box-Behnken, which were determined by representative threefactor response-surface methodology. pH and DO control was found to enhance the viable cell number of Bacilli. Most strains grew well in the optimum condition and reached nearly up to of 5×10^(9) CFU/mL. Additionally, when three of the Bacilli were cultivated in the optimized medium with pH and DO control, the viable cell number increased 3.6 times in B. subtilis, 64.1 times in B. licheniformis and 8.3 times in B. coagulans, respectively, in comparison with the original industrial medium.