To compare the effects of two radical-based extraction methods on the microstructure of Auricularia auricula polysaccharides (AAPs) and their therapeutic potential against ulcerative colitis (UC), polysaccharides were extracted using hydrogen peroxide (W-AAPs) and ascorbic acid-hydrogen peroxide (F-AAPs) methods, and their molecular weights and microstructural features were characterized. Subsequently, a dextran sulfate sodium-induced mouse model of UC was established. Mice were divided into a control group, model group, mesalazine group, and high- and low-dose W-AAPs and F-AAPs groups to evaluate the effects of polysaccharides on disease activity index, colonic histopathology, oxidative stress, inflammatory cytokines, and gut microbiota composition. The results showed that F-AAPs had a molecular weight of 87.646kDa, significantly lower than that of W-AAPs (301.89kDa). The microstructure of F-AAPs exhibited a loose rope-like network favorable for sustained utilization by beneficial gut bacteria, whereas W-AAPs formed dense lamellar structures. Compared with the model group, both 2 AAPs alleviated UC symptoms, increased serum superoxide dismutase (SOD) and glutathione peroxidase activities, decreased serum lipopolysaccharide levels, and suppressed TNF-α and IL-6 expression (P<0.05). Notably, compared with the same dose of W-AAPs, high-dose F-AAPs group exhibited superior antioxidant activity (SOD activity increased by 12.29%, P<0.05) and more pronounced anti-inflammatory effects (TNF-α decreased by 39.30%, P<0.05), which were positively correlated with the abundance of short-chain fatty acid-producing bacteria such as g_Fusicatenibacter and g_Agathobacter in the gut microbiota, particularly butyrate producers. In summary, the unique microstructure and lower molecular weight of F-AAPs contributed to its superior bioactivity in UC intervention compared with W-AAPs. These findings provided a theoretical basis for the functional development of AAPs and their potential applications in functional foods.