Abstract In the present work, a novel strategy based on overoxidized polyimidazole (PImox) and graphene oxide (GO) copolymer modified electrode was proposed for the simultaneous determination of ascorbic acid (AA), dopamine (DA), uric acid (UA), guanine (G) and adenine (A). The copolymer was characterized by the scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS). Due to the synergistic effects between PImox and GO, the proposed electrode exhibited excellent electrochemical catalytic activities and high selectivity and sensitivity toward the oxidation of AA, DA, UA, G and A. The peak separations between AA and DA, AA and UA, UA and G, and G and A were 140mV, 200mV, 380mV and 300mV, respectively. The linear response ranges for AA, DA, UA, G and A were 75–2275μM, 12–278μM, 3.6–249.6μM, 3.3–103.3μM and 9.6–215μM, respectively, and corresponding detection limits were 18μM, 0.63μM, 0.59μM, 0.48μM and 1.28μM.