Increasing demand for self-powered wearable sensors has spurred an urgent need to develop energy harvesting systems that can reliably and sufficiently power these devices. Within the last decade, reverse electrowetting-on-dielectric (REWOD)-based mechanical motion energy harvesting has been developed, where an electrolyte is modulated (repeatedly squeezed) between two dissimilar electrodes under an externally applied mechanical force to generate an AC current. In this work, we explored various combinations of electrolyte concentrations, dielectrics, and dielectric thicknesses to generate maximum output power employing REWOD energy harvester. With the objective of implementing a fully self-powered wearable sensor, a “zero applied-bias-voltage” approach was adopted. Three different concentrations of sodium chloride aqueous solutions (NaCl-0.1 M, NaCl-0.5 M, and NaCl-1.0 M) were used as electrolytes. Likewise, electrodes were fabricated with three different dielectric thicknesses (100 nm, 150 nm, and 200 nm) of Al2O3 and SiO2 with an additional layer of CYTOP for surface hydrophobicity. The REWOD energy harvester and its electrode–electrolyte layers were modeled using lumped components that include a resistor, a capacitor, and a current source representing the harvester. Without using any external bias voltage, AC current generation with a power density of 53.3 nW/cm2 was demonstrated at an external excitation frequency of 3 Hz with an optimal external load. The experimental results were analytically verified using the derived theoretical model. Superior performance of the harvester in terms of the figure-of-merit comparing previously reported works is demonstrated. The novelty of this work lies in the combination of an analytical modeling method and experimental validation that together can be used to increase the REWOD harvested power extensively without requiring any external bias voltage.