A new method of molecularly imprinted solid phase extraction (MISPE) combined with high performance liquid chromatography (HPLC) was developed for detecting five kinds of clove phenol anesthetic in aquatic product. Using eugenol as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross linking agent, molecularly imprinted polymers were synthesized by bulk polymerization and made into extraction column. Through the response surface analysis template molecule and functional monomer, crosslinking agent the best proportion was 14. 5523. 21, and the recovery rate of these compounds reached 90% by using methanol as solvent and acetic acid solution as eluent. The results showed that linear response data were obtained in the concentration range of 0.05～10 μg/mL for the five kinds of clove phenol anesthetic with correlation coefficients from 0.999 83 to 0.999 97, and the limits of quantitative were 0.05 mg/kg. The method of sample recovery rate was 82.04%～99.29% with the relative standard deviations ranging from 0.06%～3.96%. The new established method was found simple and fast，accurate and feasible, and this could be successfully applied for the determination of five kinds of clove phenol anesthetic in aquatic product and their residue with low solvent consumption.