A new process comprising hot water extraction,removal of the lipids and protease treatment was studied for the isolation of β-D-glucan from yeast cell walls in this paper.The effects of different pH,solid-liquid ratio,extraction temperature and time on extraction of β-D-glucan were investigated.The optimum process conditions were determined as following: the solid-liquid ratio,1∶15;pH,8;temperature,120℃;time,3h.The yield and purity of β-D-glucan was 61.05% and 41.51% respectively by the process.In order to improve the purity of β-D-glucan,lipid extraction and enzymatic treatment were used to remove the remaining proteins and lipids.Finally,β-D-glucan purity reached 78.31%.