The invention relates to a preparation method and application of cytochrome P450 in bacteria and belongs to the technical field of biologics. The preparation method of the cytochrome P450 in bacteria mainly comprises the following steps: a, performing freeze-crushing treatment, namely performing constant-temperature centrifugation on a bacterial liquid so as to obtain a bacterium, freezing the bacterium for 4-5 hours at minus 15 DEG C to minus 20 DEG C, and performing crushing treatment under ice-water bath so as to obtain an extract; b, performing concentration dialysis, namely concentrating the extract, and performing dialysis for 12-24 hours so as to obtain a crude product of the cytochrome P450; c, purifying, namely purifying the crude product of the cytochrome P450, thereby obtaining an enzyme finished product of the cytochrome P450. The preparation method is high in extraction rate, and the prepared cytochrome P450 is high in enzyme purity and good in activity, has an efficient degradation effect on sulfadiazine and can be used for degrading sulfonamide antibiotics.