A high performance liquid chromatographic(HPLC) method for the separation of florfenicol enantiomers using a Chiralpak AD-H(4.6 μm×250 mm，5 μm) was established.The influences of the base additive,the kinds and concentrations of alcohol additives on the chiral separation of florfenicol including resolution,retention time,theoretical plate and tailing factor were investigated.The results indicated that florfenicol was separated well from its optical isomer with n-hexane∶isopropyl alcohol∶methanol(70∶15∶15) as mobile phase at a flow rate of 1 mL?min-1 and a column temperature of 30 ℃. The UV detection wavelength was set at 224 nm.The calibration curve for florfenicol was linear in the concentration range of 0.05-0.5 g?L-1 with correlation coefficient of 0.999 7.The limit of detection(LOD) was 0.1 μg?L-1.The intra-day relative standard deviations(RSD) were less than 1.8% and the inter-day RSDs were less than 2.3%.The average spiked recoveries were in the range of 109%- 112% with RSDs not more than 3.0%.The method was reliable and simple,and was suitable for the quality control of florfenicol in industrial production.