An ultrasensitive "turn-on" fluorescent sensor was presented for determination of Hg(2+). This method is mainly based on Hg(2+)-induced conformational change of a thymine-rich single-stranded DNA. The water-soluble long-lifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as the fluorophore, which was labeled on a 33-mer thymine-rich single-stranded DNA (strand A). The gold nanoparticles (GNPs) functionalized 10-mer single-stranded DNA (strand B) is selected as the quencher to quench the fluorescence of Mn:CdS/ZnS. Without Hg(2+) in the sample solution, strands A and B could form hybrid structures, resulting in the fluorescence of Mn:CdS/ZnS being decreased sharply. When Hg(2+) is present in the sample solution, Hg(2+)-mediated base pairs induced the folding of strand A into a hairpin structure, leading to the release of GNPs-tagged strand B from the hybrid structures. The fluorescence signal is then increased obviously compared with that without Hg(2+). The sensor exhibits two linear response ranges between fluorescence intensity and Hg(2+) concentration. Meanwhile, a detection limit of 0.18 nM is estimated based on 3α/slope. Selectivity experiments reveal that the fluorescent sensor is specific for Hg(2+) even with interference by high concentrations of other metal ions. This sensor is successfully applied to determination of Hg(2+) in tap water and lake water samples. This sensor offers additional advantage to efficiently reduce background noise using long-lifetime fluorescence quantum dots by a time-gated mode. With excellent sensitivity and selectivity, this sensor is potentially suitable for monitoring of Hg(2+) in environmental applications.