Pencil graphite electrodes (PGE) were modified with over-oxidized nanofiber structured polypyrrole (PPyNF-Ox) by electropolymerization method. The performances of the pencil graphite electrodes modified with overoxidized nanofiber structured polypyrrole (PGE/PPyNF-Ox) were investigated for metronidazole (MNZ) by the differential pulse voltammetry (DPV) technique. The electrochemical signals of nanofiber and non-nanofiber electrodes for metronidazole were also determined. It was determined that over-oxidation increased the reduction current of MNZ. Surface morphology of the modified electrodes was determined by scanning electron microscopy (SEM) and electrochemical characterization was determined by cyclic voltammetry (CV). The reduction current of metronidazole was found to be diffusion-controlled. Electrochemical data showed that MNZ reduction occurred with the transfer of 4 electrons and 4 protons. The PGE/PPyNF-Ox exhibited a linear response to MNZ in the 5-500 µM concentration range. The limit of detection for PGE/PPyNF-Ox in MNZ determination was calculated as 3.8 µM and the limit of quantification was calculated as 12.8 µM. Interferent species did not significantly affect the results in metronidazole determination. The electrochemical signal obtained with the modified electrodes did not change for 20 days. In addition, the performance of these modified electrodes in real samples was tested for drug tablets containing MNZ. The optimum working pH for PGE/PPyNF-Ox is 8.0 and therefore, it offers the opportunity to work in conditions close to the pH value of body fluids.