A highly sensitive fluorescence spectroscopic method was established for the selective determination of estradiol, which took advantages of the excellent molecular recognition capability of aptamer and the energy transfer between the specific fluorescent groups. The effects of the pH value, buffer constituent and concentration, the concentration of DNA, the experimental temperature and response time on the detection of estradiol were studied. Under the optimal conditions (50 mmol/L BR buffer solution with pH value at 7. 4, 1. 0×10-7 mol/L for each DNA strand, incubation at 45 ℃, response time 19 min), the change of the fluorescence intensity (ΔI) versus the logarithm of the concentration of estradiol ( lgC) was linear over a concentration range from 1. 0×10-11 mol/L to 5. 0×10-9 mol/L with good linear correlation (r=0. 9953). The limit of detection (LOD) was found to be 6. 0×10-12 mol/L (S/N=3). This method was successfully applied to the detection of estradiol in human urine, with the recovery in the range of 94. 0%-103. 5%. This method showed good precision and accuracy.