Abstract: The purpose of this research was to extract polysaccharide from Grifola frondosa, single factor and orthogonal tests were carried out. The method of phenol-sulfuric acid was used to determine the content of polysaccharide. Antibacterial performance test about water extracts was studied, too. Orthogonal tests showed that optimum conditions of polysaccharide extraction were as follows : extraction temperature 80 ℃, liquid ratio 60∶1 (mL∶g) and extraction time 2.0h. Water extracts from G. frondosa had antibacterial activity to Bacillus subtilis, Escherichia coli and Staphylococcus aureus, and antibacterial circle were 8. 74, 10. 23, 12. 42mm respectively.