Fucose is one of important residues of recognition pattern for many immune cells. In this study, we characterized bioactive fucose-containing acidic polysaccharides from submerged fermentation of Agaricus blazei Murill. We obtained the polysaccharides through a cell-based activity-guided strategy, and used carbohydrate recognition monoclonal antibodies based Enzyme-Linked Immuno Sorbent Assay (ELISA) along with methylation and NMR analyses to investigate the structural characteristics of the polysaccharides. The polysaccharides had Mw of 3.5 × 105 Da. The major sugars were l-fucose, l-arabinose, d-galactose, d-xylose, and d-galacturonic acid in the molar ratio of 6.4, 15.5, 28.5, 14.7, and 25.0% with a small amount of d-glucose, d-mannose, l-rhamnose, and d-glucuronic acid. Results indicated that the bioactive polysaccharides consisted of a (1,4)-Galp and (1,4)-GalAp back bone; (1,2)-Xyl and (1,2)-Rha might also comprise backbone or constitute side chain; linkage (1,5)-Ara and terminal fucosyl residues were also involved in the polysaccharides. Regarding bioactivity, removal of the terminal l-fucosyl residues reduced the TNF-α cytokine stimulating activity of the polysaccharides in a RAW 264.7 macrophage cell-line test, whereas NF-κB and TLR4 affected the polysaccharide-induced TNF-α production. Display omitted •This study investigated the polysaccharide profile from submerged fermentation of Agaricus blazei Murill.•Fucose-containing acidic polysaccharides designated as F3_FA were characterized.•F3_FA polysaccharides are the major bioactive component to induce TNF-α secretion from RAW264.7 macrophages.•Terminal α-L-fucose residues play an important role of bioactivity of the polysaccharides.•F3_FA polysaccharides induced cell bioactivity affected by TLR4 receptor.