An ultra-sensitive and highly selective electrochemical label-free aptasensor is proposed for the quantitation of Hg2+ based on the hybridization/dehybridization of double-stranded DNA (dsDNA) on a gold electrode. Thiol-substituted single-stranded DNA (ssDNA) is self-assembled on the gold electrode surface through the SAu interaction. The hybridization of ssDNA with complementary DNA (cDNA) and the consequences of dehybridization in the presence of mercury ions are followed through differential pulse voltammetry (DPV) responses using a [Fe(CN)6]3−/4− redox probe. The formation of a thymine–Hg2+–thymine (T–Hg2+–T) complex is the key to producing a highly selective and sensitive aptasensor for Hg2+ determination. Specifically, the present electrochemical aptasensor is able to quantify Hg2+ ions in concentrations from 5 zeptomolar (zM) to 55 picomolar (pM) with a limit of detection of 0.6 zM, close to the dream of single atom detection, without requiring a complicated procedure or expensive materials. Keywords: Electrochemical aptasensor, T–Hg2+–T, Hg2+ detection, Zeptomolar level