The invention relates to a preparation method and an application of a petroleum degrading enzyme preparation. The preparation method comprises the following preparation steps: performing cell breakage on microorganisms for degrading petroleum to prepare crude enzyme liquid; performing mixed adsorption on the crude enzyme liquid and a carrier; and separating and drying to prepare the petroleum degrading enzyme preparation, wherein the microorganisms for degrading the petroleum are acinetobacter calcoaceticus; the collection number of the strains is CGMCC No.3915; and the collection mechanism is Common Microorganism Center of China General Microbiological Culture Collection Center. The invention also comprises the application of the prepared petroleum degrading enzyme preparation to petroleum degradation. An enzyme system of the microorganisms with a petroleum degrading function is fixed by an adsorbent and then is used for degrading soil polluted by the petroleum, so that the degradation efficiency is obviously improved and is increased by 30 to 50 times compared with the microbial degradation speed, and the stability is improved by 15 to 20 times compared with that of the crude enzyme liquid.