Given the importance of identifying the presence of biomarkers of human diseases in DNA samples, the main objective of this work was to investigate, for the first time, the electro-catalytic oxidation of 7-methyl-guanine (7-mGua) and 5-methyl-cytosine (5-mCyt) on a boron doped diamond electrode pre-treated cathodically (red-BDDE), using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The anodic peak potentials of 7-mGua and 5-mCyt by DPV were at E = 1.04 V and E = 1.37 V at pH = 4.5, indicating excellent peak separation of approximately 330 mV between species. Using DPV, experimental conditions such as supporting electrolyte, pH and influence of interferents were also investigated to develop a sensitive and selective method for individual and simultaneous quantification of these biomarkers. The analytical curves for the simultaneous quantification of 7-mGua and 5-mCyt in the acid medium (pH = 4.5) were: concentration range of 0.50–5.00 μmol L−1 (r = 0.999), detection limit of 0.27 μmol L−1 for 7-mGua; from 3.00 to 25.00 μmol L−1 (r = 0.998), with a detection limit of 1.69 μmol L−1 for 5-mCyt. A new DP voltammetric method for the simultaneous detection and quantification of biomarkers 7-mGua and 5-mCyt using a red-BDDE is proposed.