The chiral separation of zopiclone was studied by using reversed-phase high performance liquid chromatography with β-cyclodextrin（β-CD） as chiral mobile additive.Effects of some factors such as concentration of β-CD,content of methanol,pH value,flow rate and temperature on resolution were investigated.The experimental conditions were investigated by the single variable method.The best chromatographic conditions were as follows:column:Inertsil ODS-SP（5 μm,4.6 mm×250 mm）,organic phase:methanol,mobile phase:20 mmol/L phosphate buffer containing 15 mmol/L β-CD-methanol（85∶15）,flow rate:0.8 mL/min,pH value:5.5,temperature:35 ℃,detection wavelength:305 nm,injection volume:20 μL.Under the optimal conditions,the baseline separation of zopiclone enantiomers was obtained with retention time of 67.42 min and 74.48 min,and resolution of 1.62.The method is simple,sensitive and economic,and could provide a new approach for the further study of zopiclone enantiomers.