Abstract: To synthesize optically pure 3-cyclohexene-1-carboxylic acid, it was necessary to analyze the enantiomers of methyl 3-cyclohexene-1-carboxylate in actual enzymatic hydrolysis samples, the study mentioned by the title was proposed. An appropriate amount of the enzymatic hydrolysis sample solution was taken, two times of its volume of acetonitrile was added, and the mixture was mixed uniformly and then centrifuged. The supernatant was collected and passed through the 0.22 μm organic filter membrane, and the filtrate was analyzed by high performance liquid chromatography. Ultimate Cellu-JR chiral chromatographic column was used as the stationary phase, the mixed solution of acetonitrile and water at a volume ratio of 70∶30 was used as the mobile phase for isocratic elution, and detection was conducted at a wavelength of 210 nm. As shown by the results, the proposed method could be effectively used for the separation of enantiomers of methyl 3-cyclohexene-1-carboxylate. Linear relationships between mass concentrations of R-methyl 3-cyclohexene-1-carboxylate and S-methyl 3-cyclohexene-1-carboxylate and corresponding peak areas were kept in the range of 0.010‒10 g·L⁻¹, with detection limits （3S/N） of 2.10 mg·L⁻¹. Test for recovery was made by the standard addition method, giving results in the range of 94.8%‒104%, and RSDs （n=5） of the determined values obtained from intra-day and inter-day precision tests were no more than 2.0%.