Response surface methodology was employed to optimize the conditions for alkaline extraction of polysaccharides from Ganoderma lucidum. The results indicated that the optimum conditions were an extraction temperature of 60.1 degrees C, an extraction time of 77.3 min, a sodium hydroxide (NaOH) concentration of 5.1% and a substrate/liquid ratio of 1:21.4. Immunological assays results have shown that the alkaline soluble polysaccharides have no noticeable effects on monocyte phagocytosis and immune organ (spleen, thymus) weight of immunocompromised mice at the tested dosages. However, they could restore delayed type hypersensitivity reaction to dinitrofluorobenzene (DFNB), hemolysis antibody levels at the three doses applied, and improve the natural killer cell activity at the high-dose and medium dose.