Decolourization of polysaccharides is one of the crucial procedures that affects their structure, which is closely related to their bioactivity. Here, Grifola frondosa polysaccharide (GFP) was decolourized with H₂O₂ and AB-8 macroporous resin. Then, two main fractions, named DGFP and SGFP, were obtained by purification with Sepharose CL-4B. The molecular weights of these two polysaccharides were determined to be 6.306 × 10⁶ (±0.410%) Da and 1.174 × 10⁷ (±0.299%) Da by HPSEC. Monosaccharide analysis indicated that DGFP was composed of glucose, mannose, and galactose (32.20 : 1.00 : 1.75), while SGFP consisted entirely of glucose. Despite a backbone →4)-α-Glcp-(1→ in two polysaccharides, reducing ends Rα →3)-α-Glcp and Rβ →4)-β-Glcp were observed in DGFP by 1D/2D NMR. The results suggested that decolourization with low concentrations of H₂O₂ might alter the structure of GFP and generate new reducing ends. In vitro antioxidant results implied that DGFP exhibited a higher ability to scavenge DPPH and hydroxyl radicals and reduced the over-generated ROS levels in a concentration-dependent manner. These results suggested that the antioxidant effects of GFP could be activated by decolourization with H₂O₂. Therefore, DGFP might be a more promising natural antioxidant than SGFP.