A new electrode was prepared based on functionalized graphene and gold nanoparticles dispersed in a chitosan film. Such an electrochemical sensor determines ofloxacin in the presence of dopamine, paracetamol, and caffeine. Characterization (morphological and electrochemical) was done using scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. The sensor design improved the analytical signal, the electrochemical activity, and the electron transfer rate. Ofloxacin was determined by square-wave voltammetry, with a linear concentration range of 0.10-4.9 μmol L-1 (r = 0.999, LOD = 12 nmol L-1). The proposed sensor showed good repeatability and selectivity and was applied successfully to the determination of ofloxacin in pharmaceutical formulations, synthetic urine, and water river samples. The proposed method proved to be excellent; therefore, it is an alternative method for the determination of ofloxacin.