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In this paper, the development is described of an efficient pyrene excimer signaling-based fluorescent sensor for the measurement of mercury ions in aqueous solutions based on thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry and the inclusion interaction of γ-cyclodextrin. Introduction of cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of Hg2+ and enhance the fluorescence intensity of the pyrene excimer. To demonstrate the feasibility of the design, a bis-pyrene-labeled thymine-rich DNA strand was used as the detection probe. In the presence of Hg2+, stem-close-shaped DNA strands can be formed with the cooperation of γ-cyclodextrin and ideally predominantly emit the excimer fluorescence. The selectivity of the sensor for Hg2+ against other biologically and environmentally related metal ions is outstanding due to the high specificity of T–Hg2+–T formation. In addition, the pyrene excimer has a long fluorescence lifetime, which can tolerate intense background fluorescence interference from complex biological components, making it potentially applicable in the analysis of complex biological samples. [ABSTRACT FROM AUTHOR]