We report on a new type of amperometric glucose biosensor that was made by integration of glucose oxidase (GOD) with palladium nanoparticles/reduce graphene oxide (Pd/RGO) nanocomposite. The Pd/RGO was prepared by a one-step reduction method in which the palladium nanoparticles and the reduced graphene oxide (RGO) were simultaneously accomplished from the reduction of dispersed solution of PdCl2 and graphite oxide (GO) with hydrazine. The asprepared nanocomposite exhibits favorable electrocatalytic activities towards the oxidation of H2O2, which makes it a good platform for the construction of the glucose biosensor. The analytical performance of the glucose biosensor is fully evaluated. It shows good analytical properties in terms of a short response time (3 s), high sensitivity (14.1 μA/mM), and low detection limit (0.034 mM). In addition, the effects of pH value, applied potential, electroactive interference and the stability of the biosensor were discussed as well.