Abstract: The fermentation medium and conditions of Saccharomyces boulardii were optimized based on single-factor experiments and response surface method. Single-factor experiments were performed to select the optimal carbon source, nitrogen source and inorganic salt. Response surface method was used to optimize the fermentation medium and conditions. A Plackett-Burman design was used to evaluate the effects of eight factors;the path of steepest ascent and the Box-Behnken design were performed for further optimization. The optimal fermentation medium contained 2% of maltose, 2.08% of yeast extract, 2% of glucose, with initial pH value of 6.0. The optimal fermentation conditions were as follows: 5% inoculums added to 250 mL erlenmeyer flask with 29 mL medium volume were cultured with shaking speed 250 r/min at 29.6℃ for 24 h. In the optimal fermentation medium and conditions, the cell wet weight of Saccharomyces boulardii increased from 20.9 g/L to 32.2 g/L, increased by 54.1%, the number of viable yeast increased from 5.5×10⁸ CFU/mL to 8.3 ×10⁸ CFU/mL, increased by 50.9%.