A selective and highly sensitive high performance liquid chromatography (HPLC) with fluorescence derivatization method was developed for determination of ethinyl estradiol (EE); one of endocrine-disrupting compounds (EDCs). The fluorescence derivatization procedure was based on Sonogashira coupling reaction using 4-(4, 5-diphenyl-1H-imidazole-2-yl) iodobenzene (DIB-I), a fluorescence labeling reagent, to derivatize EE in presence of copper and palladium ions. The formed fluorescent product was separated on Cosmosil 5C18 MS-II by an isocratic elution with a mobile phase composed of acetonitrile: 5.0 mM Tris-HNO3 buffer, pH 7.4 (60:40, v/v %). The detection wavelengths were set at 310 and 400 nm as excitation and emission wavelengths, respectively. Various parameters affecting derivatization reaction were optimized. Further, the proposed method was validated and a good linearity with low detection limit (S/N=3) 7.4 ng L−1 was obtained in water sample after a simple solid-phase disk extraction (C18 SPE disk) method. The proposed method was successfully applied for detection of EE in river water samples in order to monitor EE concentration and to distinguish its effect on the ecosystem and human health.