In this study, PANI/MnO2 nanocomposite was synthesized by the reaction of polyaniline (PANI) and KMnO4 in the aqueous medium. Material was characterized by X-ray diffraction, nitrogen sorption, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and FT-IR. PANI/MnO2 modified electrode was investigated in the simultaneous detection of all four DNA bases (guanine, adenine, thymine, and cytosine) using cyclic and differential pulse voltammetries. pH was optimized to obtain the best peak potential separation and response current. This enabled us for the simultaneous voltammetric determination of all four DNA bases at physiological condition (pH 7). PANI/MnO2 nanocomposite exhibited high-current sensitivity for these analytes compared to MnO2 and PANI modified electrodes. Under the optimum conditions, PANI/MnO2 exhibited low detection limit, good sensitivity, and wide linear range for the simultaneous detection of G, A, T, and C. Moreover, the proposed method was successfully applied in the determination of G, A, T, and C contents in a calf thymus DNA sample with satisfactory results. The reliability and stability of the modified electrode provide a good possibility for applying the technique in the routine analysis for a selected class of electroactive organic/bio-molecules.