An ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) combined with high performance liquid chromatography-diode array detector procedure was developed for the determination of 17 beta-estradiol (beta E2), estrone (E1) and diethylstilbestrol (DES) in water. Ultrasound was applied to assist emulsification, and Triton X-100 was adopted as the disperser and emulsifier. Carbon tetrachloride was applied as the extraction solvent. Global optimization was used and the developed procedure was characterized by an acceptable linear range of 10 to 1000 ng mL(-1) for beta E2, E1 and DES (r > 0.997), a validated accuracy and precision (RSD 0.85-1.28% (n = 5)), and high sensitivity with limits of detection of 0.200, 0.100 and 0.125 ng mL(-1) for beta E2, E1 and DES, respectively. Furthermore, we applied this procedure to analyze typical water samples, with enrichment factors of 85.29, 173.45 and 97.05 for beta E2, E1 and DES, respectively, and good recoveries (>= 89.82%). Altogether, the proposed procedure was simple and reliable, with potential application in large-scale water analysis.