Based on the previous experimental results, the Box-Behnken design (BBD) was employed to study the individual and interactive effects of sucrose, malt extract, calf serum and sodium citrate on the growth of Saccharomyces boulardii, and the optimum medium compositions were obtained. Meanwhile, the optical density (OD) in the fermentation suspension was measured at 560 nm after 36 h of incubation. It is shown that the optimized medium compositions were 36.28 g·L-1 sucrose, 6.38 g·L-1 malt extract, 5.69 g·L-1 calf serum and 5.3 g·L-1 sodium citrate. The result indicated that the growth of S. boulardii could increase significantly in the optimized medium, and the OD560nm value reached 1.397 ± 0.013 after 36 h, which increased 18.59 % compared with that of pre-optimized medium. In addition, the OD560nm value 1.397 ± 0.013 in the optimized medium was very closely to the expected value 1.394. This result suggested that optimization of medium compositions for S. boulardii by BBD in this study was reliable and effective.