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A simple and rapid method was developed for the analysis of ofloxacin enantiomers by chiral ligand exchange-high performance liquid chromatography (CLE-HPLC). In addition, the influences of common cations (Ca2+, Mg2+, Fe3+, Zn2+) and the content of humic acid (HA) on the separation were investigated. The separation was carried out on a C18 column (25 cm×0.46 cm, 5 μm). The mobile phase was 20% (v/v) methanol aqueous solution containing 4 mmol/L L-isoleucine (as ligand) and 3 mmol/L CuSO4. The pH of the mobile phase was 4.5, and the flow rate was 1.0 mL/min. The column temperature was 40℃, and the detection wavelength was 293 nm. The ofloxacin enantiomers (ofloxacin and levofloxacin) were separated within 18 min and the resolution (R) was 2.70. The results showed that metal cations and HA had no significant influence on the resolution of ofloxacin enantiomers. However, the measured peak area of ofloxacin enantiomers decreased particularly in presence of Fe3+ and on increasing HA content. The proposed method can rapidly and efficiently determine ofloxacin and its chiral isomers in surface water, the influences of Fe3+ and HA content should be considered for verifying the applicability of the method.