Electrochemical DNA (E-DNA) sensors have emerged as a promising class of biosensors capable of detecting a wide range of molecular analytes (nucleic acids, proteins, small molecules, inorganic ions) without the need for exogenous reagents or wash steps. In these sensors, a binding-induced conformational change in an electrode-bound “probe” (a target-binding nucleic acid or nucleic-acid-peptide chimera) alters the location of an attached redox reporter, leading to a change in electron transfer that is typically monitored using square-wave voltammetry. Because signaling in this class of sensors relies on binding-induced changes in electron transfer rate, the signal gain of such sensors (change in signal upon the addition of saturating target) is dependent on the frequency of the square-wave potential pulse used to interrogate them, with the optimal square-wave frequency depending on the structure of the probe, the nature of the redox reporter, and other features of the sensor. Here, we show that, because it alters the driving force of the redox reaction and thus electron transfer kinetics, signal gain in this class of sensors is also strongly dependent on the amplitude of the square-wave potential pulse. Specifically, we show here that the simultaneous optimization of square-wave frequency and amplitude produces large (often more than 2-fold) increases in the signal gain of a wide range of E-DNA-type sensors.