The voltammetric behavior of 5-nitrobenzimidazole was investigated using cyclic voltammetry and differential-pulse stripping voltammetry at bare glassy carbon (GC) electrodes and DNA-modified glassy carbon (DNA-GC) electrodes. A comparison between the current responses for the 5-nitrobenzimidazole at the modified DNA-GC and unmodified GC electrodes, it was showed that the DNA- modified electrode exhibits a significant enhancement of the voltammetric current response with a better peak shape. Optimization of the pre-concentration process at the DNA-modified surface led to a significant enhancement of the voltammetric current response and a better-defined peak shape. A linear relationship between the peak current and 5-nitrobenzimidazole concentrations was observed in the range 1.0–30.0 µM, with a slope a detection limit of 0.36 µM, with r = 0.998.