A HPLC method for the determination of enantiomers in CBZ-varaciclovir was established. A Chiralcel-OD-RH-CH column(4.6 mmx150 mm, 5 μm) was used, with 90% acetonitrile(70:30) as mobile phase in sodium perchlorate solution The detection wavelength was 251 nm, column temperature was 35 °C, and the flow rate was 0.7 mL/min. For CBZ-valaciclovir isomer detection, the separation degree between the main peak and adjacent impurity peaks and known impurity peaks was >1.5, the signal-to-noise ratio S/N≥3, the response factors were basically the same, the limits of quantification were 0.012%, and the limits of detection were 0.004%. The method is simple, sensitive, reproducible, accurate and suitable for the determination of CBZ-varaciclovir isomer impurities.