We have investigated the influence exerted by the concentration of graphene oxide (GO) dispersion as a modifier for screen printed carbon electrodes (SPCEs) on the fabrication of an electrochemical biosensor to detect DNA hybridization. A new pretreatment protocol for SPCEs, involving two successive steps in order to achieve a reproducible deposition of GO, is also proposed. Aqueous GO dispersions of different concentrations (0.05, 0.1, 0.15, and 0.2 mg/mL) were first drop-cast on the SPCE substrates and then electrochemically reduced. The electrochemical properties of the modified electrodes were investigated after each modification step by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), while physicochemical characterization was performed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Finally, the sensing platform was obtained by the simple adsorption of the single-stranded DNA probe onto the electrochemically reduced GO (RGO)-modified SPCEs under optimized conditions. The hybridization was achieved by incubating the functionalized SPCEs with complementary DNA target and detected by measuring the change in the electrochemical response of [Fe(CN)6]3–/4– redox reporter in CV and EIS measurements induced by the release of the newly formed double-stranded DNA from the electrode surface. Our results showed that a higher GO concentration generated a more sensitive response towards DNA detection.