Abstract: A chiral-HPLC method was established to separate and determine the fenticonazole nitrate enantiomers.The factors such as chiral stationary phases,different proportion of mobile phase and column temperatures were investigated.Finfally,a Daicel AY-H analytical column (250 mm×4.6 mm,5 μm) was selected with the n-hexane:isopropanol:diethylamine (85∶15∶0.15) as the mobile phase at a detection wavelength of 254 nm and a flow rate of 1.0 mL/min.The column temperature was 35 °C.Under this chromatographic conditions,(-)-fenticonazole nitrate could be completely separated with its enantiomer and the resolution was not less than 1.5.The method was proved to be simple,sensitive and enantioselective,and it is suitable for determination and evaluation of fenticonazole nitrate enantiomers.