A novel one-pot chemical route for the synthesis of functional reduced graphene oxide–ZnO hybrid material (rGO–ZnO) and the development of an amperometric biosensing platform are described. The synthesis of rGO–ZnO involves the reduction of graphene oxide (GO) by zinc metal and the subsequent facile growth of ZnO over the surface of rGO using the in situ generated Zn(II) ions in aqueous solution. The ZnO nanostructures on rGO have the trigonal/tetragonal pyramidal shape with an average size of 100 nm. The hybrid material was characterized by FTIR and Raman spectroscopic, electron microscopic, XRD and electrochemical measurements. The XRD profile reveals that the ZnO has hexagonal wurtzite phase. The potential application of rGO–ZnO hybrid material in the development of amperometric biosensing platform is demonstrated with the direct electrochemistry of glucose oxidase (GOx) as a model system. GOx was immobilized on the rGO–ZnO-based electrode and direct electron transfer for the immobilized GOx was realized with a heterogeneous electron transfer rate constant of 7.55 s_1. Amperometric biosensing of glucose was achieved in the absence of oxygen at the potential of _0.3 V (Ag/AgCl). The biosensor shows a linear response from 0.2 to 6.6 mM with sensitivity and response time of 13.7 _ 0.1 mA mM_1 cm_2 and 4 s, respectively. The biosensor can detect glucose as low as 0.2 mM (S/N ¼ 3) without any interference from common coexisting electroactive species. The biosensor has excellent operational and storage stability and reproducibility. Biosensing of glucose in a small volume has been successfully demonstrated with screen printed electrodes using a human serum sample and the results are validated with clinical laboratory measurements.