In this work we report the development of an electrochemical DNA biosensor with high sensitivity for mercury ion detection. A new matrix based on gold nanoparticles (AuNPs)-glutathione (GSH)/cysteine was investigated. The interaction between DNA oligonucleotides and Hg2+ ions followed by the formation of Thymine–Hg2+–Thymine (T–Hg2+–T) structures was quantified using different electrochemical methods. It has been shown that the electrochemical impedance spectroscopy (EIS) measurements and the differential pulse voltammetry (DPV) confirmed the specific interaction between the oligonucleotide receptor layer and the Hg2+ ions. Besides, the developed sensor exhibited high sensitivity towards mercury among some examined metal ions such as Pb2+, Cu2+ and Cd2+. As a result, a high electrochemical response and low detection limit of 50 pM were estimated in the case of Hg2+ ions. The developed DNA biosensor was applied successfully to the determination of Hg2+ions in wastewater samples.