• Fabrication of hybrid electrochemical DNA biosensors via electrostatic adsorption. • In-depth optimization process of the most crucial biosensor parameters. • Large potential window allowing dsDNA cathodic/anodic processes at single electrode. • First direct monitoring of DNA damage via cathodic signals at a carbon electrode. Electrochemical DNA biosensors represent a variety of versatile analytical tools successfully used in many important applications. Their utilization in the area of monitoring DNA damage is considered as the prominent field that can provide us with an improved general knowledge regarding DNA damaging events occurring in vivo by an unorthodox overall insight employing the various electrochemical methods. Herein, we present development, optimization process, and subsequent analytical testing of the novel hybrid electrochemical DNA biosensors based on the two representatives of the pyrolytic graphite – “basal-plane” pyrolytic graphite (BPPG) and “edge-plane” pyrolytic graphite (EPPG) – and low-molecular-weight double-stranded DNA (dsDNA). Closer resolution of the nature of the oxidation/reduction signals of the dsDNA, as well as the most important optimization of parameters securing satisfying operational stability of the proposed hybrid biosensors, is investigated. Subsequent analytical testing of the novel hybrid biosensors and the evaluation of their advantages/disadvantages in terms of monitoring DNA damage caused by an UV light irradiation employing various voltammetric methods (square wave (SWV), linear sweep (LSV), or cyclic (CV) voltammetry) and electrochemical impedance spectroscopy (EIS) is also presented.