In this study, graphene oxide (GO) was directly co-deposited with polyaniline (PANI) on a Pt electrode and a 3D cluster structure of GO with wrinkled and rough surface was formed during the electro-polymerization and deposition process. Results of Raman spectra, X-ray photoelectron spectroscopy (XPS) showed that strong conjugated interactions had formed between GO clusters and PANI nanofibers. After GO was reduced, XPS measurements and electrochemical impedance spectra (EIS) studies indicated that most of the oxygen functional groups in GO were successfully removed and the conductivity of the composite material was dramatically increased. The morphology and structure of the prepared Pt/PANI/rGO/CuO electrodes were characterized by scanning electron microscopy (SEM) and the electrochemical response of the proposed electrodes in presence of glucose was investigated. Results showed that the 3D structure of rGO dusters greatly increased its effective surface area and improved the electronic transmission efficiency of the composite nanomaterial thus the Pt/PANI/rGO/CuO modified electrodes displayed much higher electrocatalytic activity than the Pt/PANI/CuO modified electrodes towards glucose, exhibiting a high sensitivity of 1252 mu A mM(-1) cm(-2), a fast response time of < 3 s, a low detection limit of 1.5 mu M (S/N = 3) and a wide linear range from 0 mM to 13 mM. The Pt/PANI/rGO/CuO electrode effectively resisted the effect of interferences such as L-ascorbic acid (AA), acetaminophen (AP) and uric acid (UA) and retained similar to 90% of its initial sensitivity after 15 days.