Chitosan (CHIT) and carbon nanofiber (CNF) modified pencil graphite electrodes (PGEs) were developed for the first time in the present study for enhanced monitoring of DNA hybridization. CHIT-CNF modified PGE, CHIT modified PGE and unmodified PGE were firstly characterized by scanning electrone microscopy (SEM), and the electrochemical behaviour of each electrode was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The optimization of experimental studies; CNF concentration, or DNA probe concentration was performed by differential pulse voltammetry (DPV) technique. The sequence-selective DNA hybridization related to Hepatitis B virus (HBV) was then performed in the case of hybridization between HBV probe and its target DNA, or different non-complementary DNA sequences (NC1 and NC2), and also the hybridization case in the mixture samples containing both target and NC sequences in the ratio of 1: 1. CHIT-CNF-PGEs presented very effective discrimination of HBV DNA hybridization, which also presented high sensitivity and better reproducibility due to the unique chemical and structural properties of CNF.