In this study, the hydrolysis conditions of β-(1, 3)-D-glucan from beer yeast were optimized by single factor and orthogonal design. The results showed that the sequence of the influential factors was as follows: sulfuric acid concentration in pretreatment hydrolysis temperature sulfuric acid concentration in hydrolysis hydrolysis time. The optimum hydrolysis conditions were as follows: 12 mol/L of sulfuric acid in pretreatment, 2 mol/L of sulfuric acid in hydrolysis, 100 oC of hydrolysis temperature and 24 h of hydrolysis time. Under these conditions,phenol-sulfuric method showed that β-(1,3)-D-glucan content reached 83.1%.