Grifola frondosa polysaccharides (GFPs) from different regions in China were characterized and compared using HPSEC-MALLS-RID and saccharide mapping based on HPAEC-PAD analysis for achieving and improving its quality control. The results showed that HPSEC chromatograms and molecular weight distributions of GFPs were similar. The average contents of each polysaccharide fraction (Peaks 1, 2, and 3) showed that Peak 3 was the main component and much higher than the other two polysaccharide fractions, which also contained protein. The result of saccharide mapping showed that α-1,4-glycosidic, β-1,4-glycosidic and few β-1,3-glycosidic linkages were existed in GFPs. The similarity result showed that HPAEC-PAD fingerprints of the oligosaccharide fragments after hydrolysis by endoglycosidase were certainly different, especially α-amylase with a mean similar index of only 0.781 ± 0.207. The result of hierarchical cluster analysis (HCA) showed that different batches of GFPs from China can be divided into different clusters. Furthermore, immune-enhancing activity based on RAW 264.7 cells showed significant differences among different GFPs. Based on grey relational analysis (GRA), the fractions of Peak 3 were regarded as the major contributors to its immuno-enhancing activity in GFPs. Overall, the implications from these results were found to be stable, comprehensive, and valid for improving the quality control of GFPs.