A ultrasound-assisted dispersive liquid-liquid microextraction based on solidification of floating organic droplet (UDLLME-SFO) combined with high performance liquid chromatography (HPLC) method was developed for the determination of quinolones (QNs) in human urine and water samples. The parameters of UDLLME-SFO procedure including sample pH, type and volume of disperser solvent, type and volume of extraction solvent and ultrasound time were optimized. The optimal extraction conditions of UDLLME-SFO were: sample pH 5; extraction solution, 300 µL 1-dodecanol; dispersion solution, 200 µL methanol; ultrasound time, 10 min. Under the optimum extraction and determination conditions, good responses for QNs were obtained in a range of 1-15 µg/mL, with linear coefficients greater than 0.97. The recoveries of the quinolones ranged from 66% to 102%. The limits of detection based on a signal-to-noise ratio of 3 was 0.03 µg/mL. The method was applied to the determination of QNs in human urine, tap and lake water samples with a satisfactory result. The results demonstrated that this UDLLME-SFO-HPLC method was successfully applied to determine QNs in urine and water samples.