This study described a physiologically relevant on-line electrochemical method for simultaneous monitoring of glucose and L-lactate in vivo with good sensitivity and selectivity. The analytical system that constructed by dual-enzyme biosensor was based on a novel and effective graphene hybrids with the on-line microdialysis system, which made the continuous and simultaneous monitoring of striatum glucose and L-lactate in brain of freely moving rats possible. The dual-enzyme biosensor was accomplished by using oxidases as the specific sensing unit and the UV-assisted synthesized Graphene/ZnO@Pd as the electrocatalyst. Transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray and X-ray photoelectron spectroscopy confirmed the denoted Pd nanoparticles (NPs) on the Graphene/ZnO composites. The analytical system showed good linearity for glucose and L-lactate concentrations from 20 mu M to 500 mu M with detection limit of 2.39 mu M and 2.52 mu M respectively. After post-calibration, the basal level concentrations of glucose and L-lactate in the microdialysate from the striatum of freely moving rats were calculated to be 0.323 +/- 0.021 mM and 0.501 mM +/- 0.057 mM (mean +/- s.d., n = 3). Moreover, the dual oxidase/Graphene/ZnO@Pd based biosensors suffered from little cross-talk and exhibit a good stability and reproducibility. (C) 2014 Published by Elsevier B.V.