A sensitive way to determine levofloxacin using a sensor based on vertical aligned carbon nanotubes is described. The morphology and the electrochemical performance of the electrodes were characterised by atomic force microscopy, cyclic voltammetry and square wave voltammetry. A scan-rate study and electrochemical impedance spectroscopy showed that the levofloxacin oxidation product is adsorbed on the electrode surface. Differential pulse voltammetry in phosphate-buffer solution allowed the development of a method to determine levofloxacin levels in the range of 1.0–10.0 µmol L−1, with a detection limit of 75.2 nmol L−1. The proposed sensor was successfully applied in the determination of levofloxacin in urine, and the obtained results were in full agreement with those from the HPLC procedure.