The invention discloses a microbial oil extraction method, and belongs to the field of bioengineering downstream technology. Oil-producing microorganism fermented liquor is used as a raw material, and is subjected to enzymolysis and extraction by organic solvent to obtain the microbial oil, wherein the extraction rate is up to 100% maximally. The microbial oil extraction method disclosed by the invention does not need concentration of the fermented liquor, recovery of strains, drying of the strains and other pretreatment operations, has mild enzymolysis conditions, short treatment time, low extraction temperature, high extraction rate, low requirements on equipment and low toxicity of the used organic solvent, greatly reduces the power and energy consumptions in the microbial oil extraction process, lowers the process cost and alleviates the environmental pollution and damage to human health. According to the method, a new way is provided for mass production and extraction of the microbial oil.