The invention discloses a preparation method of petroleum-degrading acinetobacter junii bacillus, relates to a preparation method of a petroleum-degrading bacterial agent, and is used for solving the problems that transportation and storage are inconvenient due to the fact that the petroleum is directly degraded by using the bacterial fermentation liquor, the injuries to human bodies are relatively large, and a traditional microbial agent is low in cost. The method comprises the following steps: 1, selecting acinetobacter junii, inoculating into a culture medium, and cultivating to obtain a first-stage seed solution; 2, adding the first-stage seed solution into a fermentation culture medium, and cultivating to obtain a bacterial fermentation liquor; and 3, sterilizing corncob powder, rice bran and wheat bran, mixing any two of the corncob powder, the rice bran and the wheat bran, adding the bacterial fermentation liquor, sodium chloride and monopotassium phosphate to the mixture, mixing evenly, baking, and hermetically storing, so as to obtain the petroleum-degrading acinetobacter junii bacillus. The petroleum-degrading acinetobacter junii bacillus has the advantages of being low in price, easy to degrade, and free of secondary pollution; meanwhile, the soil fertility can be improved; and the method is applied to degradation of petroleum.