A reversible and highly selective assay method has been developed to detect mercury(II) (Hg2+) ions using a conjugated polymer (CP). The transduction mechanism is based on Hg2+-mediated interpolymer pi-stacking aggregation, which results in the fluorescence self-quenching of the CP. CPs that contain thymine moieties, poly[3-(N-thymin-1-ylacetyl)ethylamine-thiophene] (PTT), have been synthesized and characteized. In the absence of Hg2+ ions, the PTT chains remain separated from each other and the CP exhibits strong fluorescence emission. Upon adding Hg2+ ions, the formation of interpolymer pi-stacking aggregation induced by specific thymine-Hg-thymine interactions results in the fluorescence quenching of PTT. Distinguishing aspects of this assay include the signal amplification of CPs and the specific binding of Hg2+ ions to thymine-thymine (T-T) base pairs.