The invention discloses a preparation method of ganoderma lucidum spores oil. The ganoderma spore powder with wall unbreaking is taken as a raw material, through steps of germination, enzymatic hydrolysis, supersonic wave freeze thawing, and extraction, the ganoderma lucidum spores oil is obtained, and the wall breaking rate can achieve more than 96%. The germination processing is carried out on the ganoderma spore powder before wall breaking, three-time freeze thawing is cooperated, the effective components in the ganoderma lucidum spores oil can be greatly kept, the wall breaking rate and oil yield of the ganoderma spore powder can be increased. The method has the advantages of simple process, low cost, high recovery rate, and stable product quality, and is suitable for industrial production.