The β-glucan was hydrolyzed by β-glucanase. The single-factor and the response surface (RSM) test were used to optimize the enzymolysis process with the yield of water-soluble yeast β-glucan as an index. The structure and thermal stability of water-soluble yeast β-glucan were examined. The results showed that the optimum conditions were substrate concentration of 1.14 g/dL, enzyme activity of 0.16 U/mL, temperature of 44 ℃, pH value of 4.2. The highest yield of water-soluble yeast β-glucan was 39.89%. The results of infrared spectroscopy showed that the glycosidic bond of water-soluble yeast β-glucan and the original yeast β-glucanwere β-configuration, and their molecular conformation were the same. The thermostability analysis showed that the characteristic decomposition temperature of water-soluble yeast β-glucan and yeast β-glucan were 356 ℃ and 360 ℃, respectively. The final decomposition temperature was 740 ℃ and 780 ℃, respectively. The difference in thermal stability between the water-soluble yeast β-glucan and the original yeast β-glucan was not significant.