Single molecule detection methods were employed to study the potential dependent adsorption and desorption of dye labeled plasmid DNA along chemical gradients prepared on indium tin oxide (ITO) electrodes. Gradients were formed over silica-base-layer-coated ITO surfaces by exposing them in a directional fashion to aminopropyltrimethoxysilane from the vapor phase. Sessile drop water contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to verify that a gradient was formed and to characterize its wettability, thickness, and composition as a function of position. The gradient-coated ITO electrode served as both the working electrode and a window into the electrochemical cell used to manipulate DNA adsorption. For single molecule studies, the electrochemical cell was filled with buffer solution containing YOYO-1-labeled plasmid DNA. Fluorescence videos acquired along the gradients depicted clear position-, potential-, and pH dependent variations in DNA adsorption and desorption. The results demonstrate that DNA adsorption was largely independent of applied potential and irreversible at high amine coverage (i.e., multilayers), under pH similar to 6 buffer. DNA adsorption became more reversible as the amine coverage decreased and the solution pH increased. Potential dependent control over DNA adsorption and desorption was best achieved at monolayer-to-submonolayer aminosilane coverage wider pH similar to 8 buffer. The knowledge gained in these studies will aid in the development of electrochemical methods for the capture and release of DNA and other polyelectrolytes at electrode surfaces.