Poly-L-lysine is one of the biocompatible polymers having amino and carboxyl groups in its structure. This attractive feature of poly-L-lysine makes it very convenient for bioactive molecule attachment. This study details the preparation of poly-L-lysine-based pencil graphite electrodes (PLL/PGEs) and use of the coated electrodes for direct ultrasensitive DNA hybridization detection. In the first part of this study, poly-L-lysine coated electrodes were prepared using L-lysine as the monomer by cyclic voltammetry (CV) with different cyclic scans. The effect of these cyclic scans during the electropolymerization was investigated. Coated electrodes were characterized by cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Then, one-pot preparation of poly-L-lysine composites with graphene (GN) and multi-walled carbon nanotubes (MWCNTs) onto the pencil graphite electrodes were achieved. Electrochemical responses of these 3 electrodes were compared. After all, electrochemical DNA hybridization was performed using the poly-L-lysine-based electrodes prepared at optimum polymerization condition. The PLL/PGE coated electrode presented a good linear response in the target concentration range of 1.0x10(-13) to 1.0x10(-6) with a detection limit of 2.25x10(-14) using differential pulse voltammetry as the detection method. We believe that poly-L-lysine-based surfaces will be useful for further clinical applications.