A novel method was developed for the synergetic accumulation and simultaneous determination of naphthol isomers (alpha-N and beta-N) at electrochemically reduced graphene oxide modified glassy carbon electrode (E-rGO/GCE). One-step electrochemical reduction was employed for the fabrication of E-rGO/GCE, then the modified material was characterized by scanning electron microscope, Raman spectroscopy and Fourier transform infrared spectroscopy. Electrochemical behavior of naphthol isomers at E-rGO/GCE shows that the sensitivity of the isomers could be increased due to the synergistic effect of n-rt conjugating and electrochemical accumulation. The oxidation peaks of a-and (3 -naphthol could be completely separated and the isomers were simultaneously detected with the linear ranges of 5-400 nM and 0.5-20 IN for a-N, 5-350 nM and 0.5-2011M for p-N. The detection limits of alpha-N and beta-N were 1.01 nM and 0.43 nM (S/N = 3), and the corresponding sensitivity were 0.0097 mu A/mu M and 0.0113 mu A/mu M, respectively. More importantly, the naphthol isomers electrochemical sensor on E-rGO film with synergistic effect can be expected to result in significant improvements in the sensor performance and the developed method has been successfully applied to human urine sample with a satisfactory result. (C) 2016 Elsevier B.V. All rights reserved.