A new method for determination of β-D-glucans content in Lentinan was developed by acidic and enzymatic hydrolysis coupled with high performance liquid chromatography(HPLC).Lentinan was hydrolyzed entirely to glucose with exo-1,3-β-glucanase and β-Glucosidase after partial acid hydrolysis for determination of total glucans by HPLC.α-Glucans in Lentinan samples were hydrolyzed to glucose with Amyloglucosidase and then also analyzed by HPLC.The content of β-D-glucan was calculated by the difference between total glucans and α-glucans.Moreover,the above method and phenol sulfuric acid method and Congo red method were used respectively to determine the content of glucans(or β-D-glucan) in laminarin,curdlan and yeast glucan samples whose β-D-glucan content were known.Compared the results from three methods,acidic and enzymatic hydrolysis coupled HPLC method were the most suitable for quantitative analysis of β-D-glucans in Lentinan.RSD of the method were 2.02%～2.94%.