Here we investigate a novel signal-on electrochemical DNA sensor based on the use of a clamp-like DNA probe that binds a complementary target sequence through two distinct and sequential events, which lead to the formation of a triplex DNA structure. We demonstrate that this target-binding mechanism can improve both the affinity and specificity of recognition as opposed to classic probes solely based on Watson-Crick recognition. By using electrochemical signaling to report the conformational change, we demonstrate a signal-on E-DNA sensor with up to 400% signal gain upon target binding. Moreover, we were able to detect with nanomolar affinity a perfectly matched target as short as 10 bases (K(D) = 0.39 nM). Finally, thanks to the molecular "double-check" provided by the concomitant Watson-Crick and Hoogsteen base pairings involved in target recognition, our sensor provides excellent discrimination efficiency toward a single-base mismatched target.