Macrofungi, a class of unique natural resources, are gaining popularity owing to their potential therapeutic benefits and edibility. From Fomitopsis officinalis, a medicinal macrofungus with anticancer activity, a homogeneous heteropolysaccharide (FOBP50–1) with a molecular weight of 2.21 × 104 g/mol has been extracted and purified. FOBP50–1 was found to be composed of 3-O-methylfucose, fucose, mannose, glucose, and galactose with a ratio of 1: 6.5: 4.4: 8.1: 18.2. The sugar fragments and structure of FOBP50–1 were investigated, which included →6)-α-d-Galp-(1→, →2,6)-α-d-Galp-(1→, →3)-α-l-Fucp-(1→, α-d-Glcp-(1→, →3)-β-d-Manp-(1→, →6)-β-d-Manp-(1→, 3-O-Me-α-l-Fucp-(1→, according to the UV, FT-IR, GC–MS, and NMR data. Besides the structure elucidation, FOBP50–1 showed promising antitumor activity in the zebrafish assays. The following mechanism examination discovered that FOBP50–1 interacted with TLR-4, PD-1, and VEGF to activate immunity and inhibit angiogenesis according to a series of cell, transgenic zebrafish, and surface plasmon resonance (SPR) experiments. The KD values indicating the association of FOBP50–1 with TLR-4, PD-1, and VEGF, were 4.69 × 10−5, 7.98 × 10−6, 3.04 × 10−6 M, respectively, in the SPR experiments. All investigations have demonstrated that the homogenous fungal polysaccharide FOBP50–1 has the potential to be turned into a tumor immunotherapy agent.