We have developed a technique for the highly selective and sensitive detection of Pb(2+) and Hg(2+) using a thrombin-binding aptamer (TBA) probe labeled with the donor carboxyfluorescein (FAM) and the quencher 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) at its 5' and 3' termini, respectively. The TBA has a random coil structure that changes into a G-quartet structure and a hairpin-like structure upon binding Pb(2+) and Hg(2+) ions, respectively. As a result, the fluorescence decreases through fluorescence resonance energy transfer (FRET) between the fluorophore and quencher. These changes in fluorescence intensity allow the selective detection of Pb(2+) and Hg(2+) ions at concentrations as low as 300 pM and 5.0 nM using this TBA probe in the presence of phytic acid and a random DNA/NaCN mixture, respectively. The linear correlation existed between the fluorescence intensity and the concentration of Pb(2+) and Hg(2+) over the range of 0.5-30 nM (R(2) = 0.98) and 10-200 nM (R(2) = 0.98), respectively. To the best of our knowledge, this is the first example of a single DNA-based sensor that allows the detection of both Hg(2+) and Pb(2+) ions. This simple and cost-effective probe was also applied to separately determine Pb(2+) in soil samples and spiked Hg(2+) in pond samples.