In this study, a highly sensitive, electrochemical, and label-free DNA impedimetric sensor was developed using carbonized glass fiber–coal tar pitch (GF–CTP) electrodes supported with gold nanoparticles (AuNPs) for the detection of HIV-1 gene. Thiol-modified GF–CTP electrodes were prepared using amine crosslinking chemistry and AuNPs were self-assembled obtaining highly conductive nanoelectrodes, GF–CTP–ATP–Au. All steps of electrode modifications were characterized using electrochemical, spectroscopic, and microscopic methods. GF–CTP–ATP–Au electrode was then modified with a capture DNA probe (C-ssDNA) and optimized with a target DNA probe in terms of hybridization time and temperature between 30 and 180 min and 20–50 °C, respectively. Finally, the analytic performance of the developed ssDNA biosensor was evaluated using electrochemical impedance spectroscopy. The calibration of the sensor was obtained between 0.1 pM and 10 nM analyte working range. The limit of detection was calculated using signal to noise ratio of 3 (S/N = 3) as 13 fM. Moreover, interference results for two noncomplementary DNA probes were also tested to demonstrate non-specific ssDNA interactions. An electrochemical label-free DNA impedimetric sensor was successfully developed using a novel GF–CTP–ATP–Au electrode. This study suggests that highly sensitive DNA-based biosensors can be developed using relatively low-cost carbonaceous materials.