A novel turn-on Hg²⁺ sensor was constructed based on fluorescent C–Hg²⁺-aptamer DNA-stabilized Ag nanoclusters (DNA–AgNCs), and was used to determine the concentration of Hg²⁺ over the range 2–18 nM with a detection limit as low as 0.25 nM. The sensing assay relied on a target-induced conformational transition of Hg²⁺-aptamer DNA. The conformational change of Hg²⁺-aptamer DNA from a single strand to a hairpin DNA due to the formation of a T–Hg²⁺–T complex in the presence of Hg²⁺ made the two darkish DNA–AgNCs approach each other closely and then enhanced the fluorescence of the AgNCs, which enabled the sensitive and specific detection of Hg²⁺. The proposed sensor was found to be easy to use, and allowed for the sensitive, selective, and turn-on detection of Hg²⁺. Furthermore, this approach has also been successfully applied to the detection of Hg²⁺ in real water samples, so this sensor may find application in monitoring Hg²⁺ in environmental samples.