Herein we report a simple, one-step approach to prepare a low-cost and binder free MoS 2 -pencil graphite electrode (i.e., MoS 2 -PGE) for the electrochemical oxidation of DNA nucleobases i.e., guanine (G) and adenine (A) in physiological pH (7.4) buffer solution. MoS 2 -PGE was synthesised by hydrothermal method and the morphology of such hybrid was characterized by field emission scanning electron microscopy, X-ray diffraction, Raman and X-ray photoelectron spectroscopy. In cyclic voltammetry, MoS 2 -PGE displays two well-seprated and well-defined irresversible peaks at 0.58 and 0.90 V for electrochemical oxidation of G and A respectively when compared to bare PGE. Likewise, differential pulse voltammetry of MoS 2 -PGE showed well-seprated and sharp peak current responses for G and A at 0.56 V and 0.85 V respectively. Under optimized conditions, DPV was further adopted for simultaneous and separation-free determination of G and A in physiological pH. MoS 2 -PGE shows good stability with linear range of 15–120 μM and 15–120 μM for G and A detection respectively. Obtained sensitivity and limit of detection (signal-to-noise = 3) are comparable with the previous literature. As an immediate practical applicability, MoS 2 -PGE was used for quantification of G and A concentration in calf-thymus DNA and detected ratio of G and A (i.e., [G]/[A]) ratio is 0.85. The current approach provides a new avenue towards the development of affordable electrodes for a wide range of bioanalytical applications. © 2018