A Cu2O/NiOx/graphene oxide (GO) modified glassy carbon electrode (GC) was prepared by electrodeposition successively. In this layer-by-layer structure, monodisperse and uniform spherical or octahedral Cu2O could be synthesized by controlling the electrodeposition potential, and three-dimensional NiOx nanoparticles was electrodeposited on the surface of GO. The presented electrode exhibited high elctrocatalytic activity toward the oxidation of reduced glutathione (GSH) and glucose due to the synergistic effect of Cu2O and NiOx which was applied in the electrochemical determination of GSH at physiological pH and nonenzyme glucose sensor. The results showed that, with the co-effect of three-dimensional NiOx nanoparticles, spherical Cu2O demonstrated much higher electrocatalytic activity than octahedral Cu2O. In addition, interference from the oxidation of common interfering species can be avoided. (C) 2013 Elsevier Ltd. All rights reserved.