Required routine monitoring of endocrine disrupting compounds (EDCs) in water samples, as posed by EPA Unregulated Contaminant Regulation (UCMR3), demands for cost-effective, reliable and sensitive EDC detection methods. This study reports a reusable evanescent wave aptamer-based biosensor for rapid, sensitive and highly selective detection of 17β-estradiol, an EDC that is frequently detected in environmental water samples. In this system, the capture molecular, β-estradiol 6-(O-carboxy-methyl)oxime-BSA, was covalently immobilized onto the optical fiber sensor surface. With an indirect competitive detection mode, samples containing different concentrations of 17β-estradiol were premixed with a given concentration of fluorescence-labeled DNA aptamer, which highly specifically binds to 17β-estradiol. Then, the sample mixture is pumped to the sensor surface, and a higher concentration of 17β-estradiol leads to less fluorescence-labeled DNA aptamer bound to the sensor surface and thus to lower fluorescence signal. The dose-response curve of 17β-estradiol was established and a detection limit was determined as 2.1 nM (0.6 ng mL(-1)). The high specificity and selectivity of the sensor were demonstrated by evaluating its response to a number of potentially interfering EDCs. Potential interference of real environmental sample matrix was assessed by spiked samples in several tertiary wastewater effluents. The sensor can be regenerated with a 0.5% SDS solution (pH 1.9) over tens of times without significant deterioration of the sensor performance. This portable sensor system can be potentially applied for on-site real-time inexpensive and easy-to-use monitoring of 17β-estradiol in environmental samples such as effluents or water bodies.