The invention relates to a preparation method of a molecularly imprinted polymer microsphere in the technical field of the chemical engineering. The invention includes the following steps: under the effect of ultrasound, the mixture is added in the solution and quickly stirred to react for 24 hours; after stirring, filtering and washing, the template molecules are removed by the soxhlet extraction; the polymer removed template molecules are dried to constant weight in vacuum, and the molecularly imprinted polymer microsphere is obtained. The method of separating enrofloxacin from the molecularly imprinted polymer microsphere includes the steps that the molecularly imprinted polymer microsphere of enrofloxacin is filled into the polypropylene solid phase extraction cartridge; the solid phase extraction cartridge is activated and decontaminated, and finally, the enrofloxacin for detecting is eluted and collected. With simple method and perfect mechanical intensity and chemical stability,the invention can be used for the extreme conditions such as acid, alkali, high temperature, etc., and is also applicable to separate, purify and enrich the enrofloxacin in such complex substrates aswater-solubility medium and animal-originated food, thereby possessing a perfect application prospect.