 The carboxylic acid-functionalized graphene oxide/gold nanoparticles modified glassy carbon electrode has been utilized as a platform to immobilize 5-amino-2-hydroxybenzoic acid (mesalazine). The surface structure and composition of the sensor were characterized by scanning electron microscopy. Electrocatalytic oxidation of urea on the surface of modified electrode was investigated with cyclic voltammetry, electrochemical impedance spectroscopy, and hydrodynamic voltammetry methods. The cyclic voltammetric results indicated the ability of carboxylic acid-functionalized graphene oxide gold nanoparticles modified glassy carbon electrode to catalyze the oxidation of urea. In addition, the modified electrode has short response time, low detection limit, high sensitivity, and low operation potential. Some kinetic parameters, such as the electron transfer coefficient, diffusion coefficient, and catalytic rate constant of the catalytic reaction were calculated. A sensitive amperometric method was proposed for determination of urea with advantages of fast response and good reproducibility.