Abstract: The present invention relates to a method for preparing Ganoderma spore oil which is in the field of biotechnology. The method includes using Ganoderma spore powder and Ganoderma powder (obtained from grinding fruiting bodies) as raw materials, applying enzymatic Ganoderma sporoderm broken methods, one-step granulation, supercritical CO2 extraction method, as well as centrifugation and refining. The light yellow oleaginous substances obtained posses a variety of physiological functions such as strengthening immunity, protecting the liver and inhibiting tumor cell growth, etc. Ganoderma spore oil prepared with the present technology contains not only spore extracts, but also extracts from Ganoderma fruiting bodies and mycelium by CO2 supercritical extraction, with more types of triterpenoids. The inhibitory effects of Ganoderma spore oil is a onefold higher than spore oil prepared from the physical preparation of the sporoderm-broken spore.