For the first time, both theoretical and electrochemical behavior of the phenylurea herbicide fluometuron (FTN) were investigated using the glassy carbon electrode (GCE). The cyclic voltammetry (CV) performed in the presence of FTN showed an irreversible profile for the analyte with one well-defined anodic peak at ca. +1.2 V (vs. Ag/AgCl). The linear dependence of pH and peak potential was evaluated using the Nernst equation through CV measurements. The slope of 46 mV s−1 suggests an oxidation mechanism involving an equal number of protons and electrons. High-level quantum-mechanical calculations based on the density functional theory showed that the presence of defects on the GCE surface induces strong interaction leading to FTN dissociation and/or its adsorption with orientation parallel to the substrate which compromises the electroanalysis. Raman spectroscopy confirmed the surface fouling. Differential pulse voltammetry (DPV) was used for the electroanalytical determination of FTN under optimized conditions. The oxidation peak increased with increasing concentrations of FTN in the range of 207–3846 µg L−1; the theoretical detection limit was estimated as 63 µg L−1. To avoid surface fouling, the calibration curve was applied for the determination of FTN. The unmodified electrode offered proper selectivity in the presence of other pesticides and potential interfering species. Intra and inter-day precision was established. Recoveries were obtained in the range of 81.8–98.9% (n = 3) with the absence of matrix effects. The results showed that electroanalysis through the bare GCE might be used as a novel strategy to determine the persistent organic compound FTN in spiked river water and simulated serum samples and preliminary evaluations such as screening purposes.