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In this paper, we developed a fluorescent aptasensor for 17β-estradiol (E2) determination in aqueous solution using label-free E2-specific aptamer, gold nanoparticles (AuNPs) and Rhodamine B (RhoB) as sensing probe, fluorescent quencher and fluorescent indicator respectively. In the absence of E2, AuNPs were wrapped by E2 aptamer and maintained dispersed in NaCl solution basically. These dispersed AuNPs could effectively impair the originally high fluorescence of RhoB. Contrarily, in the presence of E2, E2 aptamer could specifically combine with E2 to form E2-aptamer complex, so the AuNPs were released by E2 aptamer and aggregated under the influence of NaCl. The aggregated AuNPs have a weak influence on RhoB fluorescence. Therefore, the E2 concentration can be determined by the change of fluorescence intensity of RhoB. This fluorescent assay has a detection limit as low as 0.48 nM, a linear range from 0.48 to 200 nM, and high selectivity over other disrupting chemicals. It was applied to determine E2 in water samples with recoveries in the range of 94.3–111.7%. The fluorescent aptasensor holds great potential for E2 detection in environmental water samples. [ABSTRACT FROM AUTHOR]