We report a signal-on, reagentless electrochemical DNA biosensor, based on an electroactive self-assembled naphthoquinone derivative (JUG(thio)) monolayer. This system achieves highly sensitive (similar to 300 pM) and selective signal-on detection. Before hybridization, the single strand can interact with JUG(thio) and slow down the redox reaction. When the complementary target is added, the formation of the double helix eliminates the single strand/JUG(thio) interactions and the JUG(thio) redox rate, and hence the current increase.