Coupling T base with Hg²⁺ to form stable T–Hg²⁺–T complexes represents a new direction in detection of Hg²⁺. Here a graphene oxide (GO)-based fluorescence Hg²⁺ analysis using DNA duplexes of poly(dT) that allows rapid, sensitive, and selective detection is first reported. The Hg²⁺-induced T₁₅–(Hg²⁺)_n–T₁₅ duplexes make T₁₅ unable to hybridize with its complementary A₁₅ labelled with 6′-carboxyfluorescein (FAM-A₁₅), which has low fluorescence in the presence of GO. On the contrary, when T₁₅ hybridizes with FAM-A₁₅ to form double-stranded DNA because of the absence of Hg²⁺, the fluorescence largely remains in the presence of GO. A linear range from 10 nM to 2.0 μM (R² = 0.9963) and a detection limit of 0.5 nM for Hg²⁺ were obtained under optimal experimental conditions. Other metal ions, such as Al³⁺, Ag⁺, Ca²⁺, Ba²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Co²⁺, Pb²⁺, Ni²⁺, Cu²⁺, Cd²⁺, Cr³⁺, Fe²⁺, and Fe³⁺, had no significant effect on Hg²⁺ detection. Moreover, the sensing system was used for the determination of Hg²⁺ in river water samples with satisfactory results.