The invention relates to a method for preparing fly oil and relative fatty acid, which comprises that washing the fly water, under 0.01MPa, drying it until the water content of fly is lower than 15%, breaking, adding organic solvent, extracting, separating solid and liquid, recycling the liquid to recycle the solvent to obtain the fly oil; in the buffer liquid prepared from sodium hydrogen phosphate and acid sodium phosphate at 6.0-8.0pH, adding neutral lipase, to reach 180-200 lipase active amount in each milliliter; adding fly oil into the buffer liquid at 1:0.5-1.5 volume ratio, mixing, reacting, stopping reacting, laying and separating, separating the oil, washing with the water in same volume, rotating and drying the oil, to obtain the fly fatty acid. The inventive fly oil and relative fatty acid can be used to treat liver cancer, lung cancer, or the like.