Abstract: To determine the concent of alkali- insoluble yeast cell wall polysaccharides,the solution of H2SO4 was used to hydrolysis the alkali- insoluble yeast cell wall polysaccharides,then the saccharide of the hydrolytic solution was determined by phenol- sulfuric acid method. The hydrolysis condition was optimized by single factor and orthogonal experiments.The optimized hydrolysis condition was as follows: 10 mg alkali- insoluble yeast cell wall polysaccharides were hydrolyzed for 90 min at 100℃ with 20 m L 3mol / L H2SO4 solution. Then the solution was volumed to 250 m L and the content of saccharide was determined. The RSD of precision was 0.30%. The RSD of stability was 0.67%. The RSD of reproducibility was 0.29%. The average spike recovery was 99.87%,with 1.16%RSD.The method was effective for determination of alkali- insoluble yeast cell wall polysaccharides.