Abstract: A fluorescence aptasensor for quantitative detection of 17β-estradiol was constructed. Black phosphorus nanosheets (BPNs) were prepared by ultrasound-assisted aqueous phase exfoliation using black phosphorus crystal (BP) as fluorescence receptors. A 17β-estradiol fluorescence aptasensor was constructed between FAM labeled aptamers and BPNs based on the fluorescence resonance energy transfer (FRET). After optimization of BPNs concentration, FAM-Apt concentration and reaction time, samples of tap water and milk were determined. The results showed that the prepared BPNs had independent lamellar structure and uniform particle size.With the concentrations of BPNs and FAM-Apt were 10 μg/mL and 7.5 nmol/L respectively, the linear range of the sensor was 1.5~60 ng/mL and the detection limit was 1.0 ng/mL. The recoveries of tap water and milk were 91.2%~104.8% and 87.5%~104.3%. The relative standard deviation (RSD) were 4.99%~10.53% and 4.48%~11.24%, respectively. This method with simple, high sensitive and strong specificity could be completed within 30 min and could realize quantitative detection of E2 in actual samples.