A dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) combined with ultraviolet spectrometry (UV) method was developed for the determination of diethylstilbestrol (DES) in water samples. Several parameters including sample pH, extraction solution type and volume, dispersion solution type and volume, centrifugation speed and time were systematically investigated. The optimal extraction conditions of DLLME-SFO were: sample pH 3; extraction solution, 200 μL 1-dodecanol; dispersion solution, 400 μL ACN; Centrifugation speed and time, 2500 rpm and 15 min . Under the optimum extraction and determination conditions, good responses for DES were obtained in a range of 0.5 - 12.5 μg/mL, with linear coefficients greater than 0.99. The recoveries of the DES ranged from 70% to 97%. The limits of detection based on a signal-to-noise ratio of 3 was 0.15 μg/mL. The method was applied to the determination of DES in tap and lake water samples with a satisfactory result. The results demonstrated that this DLLME-SFO-UV method was successfully applied to determine DES in water samples.