In this paper, combined Hg²⁺ specific recognition of thymine with chronocoulometry, we constructed a highly sensitive electrochemical biosensor based on DNA-mediated charge transport for the detection of Hg²⁺ in aqueous solution. DNA duplexs with one T-T base mismatch were assembled onto a gold electrode surface through Au-S bond. Chronocoulometry was used to detect Hg²⁺ in 2 mmol/L[Fe(CN)₆]^3- (0.1 M KCl, pH 7.4) containing 2.0 μmol/L MB. T-T mismatch blocked the internal charge transfer of the DNA duplexs. In the presence of Hg²⁺, the DNA on the electrode surface specifically recognized Hg²⁺ and formed thymine-Hg²⁺-thymine complexs. Thus, internal charge transfer path in dsDNA was formed, significantly improving charge transport onto the gold electrode surface. The results showed the chronocoulometry of DNA-modified gold electrode increased with the increase of Hg²⁺ concentration at the reduction peak of methylene blue (-380 mV). The change of chronocoulometry was linear with regard to lgc_Hg²⁺ over a concentration range from 1.0 n to 10⁴ nmol/L (R²=0.997) and with a detection limit of 0.5 nmol/L (S/N=3). A test for a series of interference metal ions showed that this biosensor based on DNA-mediated charge transport is highly specific and selective toward Hg²⁺.