A novel aptamers-based fluorescence resonance energy transfer (FRET) assay was developed employing the oppositely charged thioglycolic acid-capped CdTe quantum dots (TGA-CdTe QDs) and cysteamine-stabilized gold nanoparticles (cysteamine-AuNPs). The fluorescence of TGA-CdTe QDs was significantly quenched with addition of cysteamine-AuNPs via the FRET. The FRET process can be modulated by 17β-estradiol in that the specific recognition between 17β-estradiol and aptamers could show different effects on the aptamers-mediated aggregation of cysteamine-AuNPs, and correspondingly adjust the FRET process. The feasibility of the method has been demonstrated by carrying out the detection of 17β-estradiol with the wide linear range from 0.5·ng mL-1 to 150 ng·mL-1 and the low detection limit of 0.057 ng·mL-1. The established assay exhibited favorable selectivity towards 17β-estradiol over other endocrine disrupting compounds and probably coexisting chemicals in real samples. Furthermore, the assay has been successfully applied to detect 17β-estradiol in real tap water samples and feeds samples with good performance. The results were in full consistence with those from HPLC method, indicating the reliability of the detection system. The aptamers-based FRET assay is expected to offer a new opportunity for the rapid analysis of 17β-estradiol in real samples.