A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for simultaneous enantiomeric analysis of flumequine and its metabolite 7-hydroxyflumequine in water and sediment had been developed based on the separation method. Sediment samples were extracted with ACN and EDTA-Mcllvaine buffer solution (40:60, v/v) then were enriched and cleaned-up by Cleanert PEP solid-phase extraction cartridges. The extract solvent, solid cartridges, mobile phase ratios, and chiral separation column were all optimized to reach high sensitivity and selectivity, good peak shape, and satisfactory resolution. The results showed that the calibration curves of flumequine enantiomers and 7-hydroxyflumequine were linear in the range of 1.0 to 200.0 µg/L with correlation coefficients of 0.9822-0.9988, the mean recoveries for both the enantiomers ranged from 69.9-84.6% with relative standard deviations (RSDs) being 13.1% or below. The limits of detection (LODs) for both flumequine enantiomers were 2.5 µg/L and 5.0 µg/kg in water and sediment samples, whereas the limits of quantification (LOQs) were 8.0 µg/L and 15.0 µg/kg, respectively. While the LODs for 7-hydroxyflumequine were 3.2 µg/L in water samples and 7.0 µg/kg in sediment samples. The proposed method will be extended for studies on the degradation kinetics and environmental behaviors and providing additional information for reliable risk assessment of these chiral antibiotics.