Saccharomyces boulardii as the probiotic yeast was widely used in the pharmaceutical, feed and food industries. The influence of skim milk, gelatin, and carbohydrates on the heat resistance of S. boulardii is explored in the article. Response surface methodology was effectively applied to optimize the thermoprotectant composition for S. boulardii during spray-drying. The accelerated test is applied to evaluate its the subsequent storage stability. The results show that the thermoprotectants composition was comprehensively optimized such as: 15.12% skim milk, 1.81% gelatin, and 9.73% trehalose. The highest viability was 17.77%, which was basically the same as the predicted value of 18.21%. The inactivation rate constant of spray-dried powder was k-18 = 1.04 × 10-5 h-1, the quantity of viable cells stored at this temperature for 1 and 10 years was 8.25 × 108 CFU/g and 1.25 × 108 CFU/g, separately. This work provides a thermoprotectants formula for the S. boulardii during the spray drying process.