A novel “signal-on” assay for sensitive and selective detection of Hg2+ in aqueous solution based on mercury-specific DNA and a molecular light switch complex, SYBR GREEN I, was reported in the present work. This Hg2+ specific sensor system composed of two labelfree DNA probes with five T-T mis-matches, which could form stable DNA duplexes by thymine-Hg2+-thymine(T-Hg2+-T)in the presence of Hg2+. The fluorescence of SYBR GREEN I is weak in aqueous solution, and significant fluorescence can be observed when intercalating into DNA duplexes. By monitoring Hg2+-dependent fluorescence intensity enhancement, highly sensitive and selective determination of Hg2+ has been achieved. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of Hg2+ in the range of 5-100 nmol/L. A detection limit of 2 nmol/L Hg2+ was achieved. The presence of other metal ions did not interfere with the detection of Hg2+. This approach is simple and rapid, which can be used to monitor the Hg2+ concentration in drinking water.