An electrochemical biosensor was developed for Hg2+ determination based on DNA hybridization. In the presence of Hg2+, the target and probe DNAs with thymine - thymine (T-T) mismatches could hybridize by forming T-Hg2+-T complex. This induced DNA hybridization led to the decrease in reduction peak currents of ethyl green (EG) as electroactive label, which could be used for determination of Hg2+. The difference in the value of the peak currents of EG before and after DNA hybridization (∆I) was linear with the concentration of Hg2+ in the range of 9.0×10-11 - 1.0×10-9 M. The detection limit was 3.08×10-11 M.