A graphene oxide (GO) modified glassy carbon electrode (GCE), namely GO/GCE was prepared by covalent coupling method, which was characterized by atomic force microscope (AFM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS). On this modified electrode, it is found that the electrochemistry of dopamine (DA) is greatly enhanced, while that of ascorbic acid (AA) is totally impressed, showing that the modified layer of GO has completely different impact on the electrochemical response of DA and AA. The probable mechanism to cause the different impact was proposed. GO/GCE was further applied as a biosensor for the determination of DA in the presence of with AA, and the results showed that the coexisted AA has no interference toward the electrochemistry of DA. The oxidation peak currents of DA present a good linear relationship with the concentrations in the range from 1.0 μM to 15.0 μM with a detection limit of 0.27 μM. The electrochemical parameters such as the electron transfer rate constant, catalytic rate constant, diffusion coefficient, and electron/proton transfer number of DA on GO/GCE were also studied.