The invention discloses a preparation method of milbemycin oxime. The preparation method comprises the following steps: taking an ethyl acetate-ethanol solution as a reaction solvent to dissolve milbemycin ketone, and adding and a water solution of hydroxylamine hydrochloride for carrying out a reaction for 4-6 hours to prepare the milbemycin oxime. The preparation method also comprises the stepsof purifying the prepared milbemycin oxime, and the specific purifying steps are as the follows: washing the prepared milbemycin oxime with water, dissolving the washed milbemycin oxime with ethanol,dropwise adding the obtained solution into water for crystallization under stirring, and carrying out filtering and drying to obtain the pure product of milbemycin oxime. Compared with the prior art,the preparation method can be used to greatly reduce the occupational health hazard of employees by using the low-toxicity solvent instead of a high-toxicity solvent. The reaction time is greatly shortened, and the production efficiency is improved.