Four polysaccharide fractions, LRP-1, LRP-2, LRP-3 and LRP-4 were extracted stepwise from Lignosus rhinocerotis sclerotia with distilled water at 25, 95, 120 °C and 1.0 M NaOH solution at 4 °C. Their structure, molecular size and chain conformation were clarified using SEC-MALLS-RI, GC, FT-IR and UV–vis. Furthermore, their immunomodulatory activities were evaluated by the model of cyclophosphamide (Cy)-induced immunosuppression. The LRP-1 and LRP-2 were polysaccharide-protein complexes (46–68% β-d-glucan and 27–48% protein), while LRP-3 and LRP-4 were absolutely composed of β-d-glucose. The LRP-4 with low polydispersity had much higher molecular weight (M_w, 5.86 × 10⁶ g/mol) and intrinsic viscosity ([η], 202.6 ml/g) than other LRP fractions. Based on M_w, radius of gyration (<S²>_z^1/2) and [η] data with the exponent β of <S²>_z^1/2–M_w and its U-shaped curve, all four LRP fractions were highly branched macromolecules and LRP-3 showed a more compact sphere-like conformation than LRP-2 in aqueous solution. Additionally, all four LRP fractions exhibited protective effects against Cy-induced immunosuppression in mice by improving immune organs as well as stimulating the release of major cytokines TNF-α and INF-γ. This work provides a theoretical basis for the application of polysaccharides and their protein complexes from Lignosus rhinocerotis sclerotia in food- or drug-based therapies.