In this study, we used a facile approach to decorate Ni nanoparticles (Ni-NPs) with ethylene glycol followed by their adsorption on reduced graphene oxide (rGO) nanosheets for the development of enzyme free electrochemical glucose sensor. The effect of Ni-NP content (0.14, 0.28, 0.42, and 0.56 wt.%) on electrochemical properties and the molecular interaction of glucose with Ni-NPs/rGO in glucose sensing were evaluated. Ni-NPs/rGO showed significantly higher electrochemical performance for the glucose oxidation in alkaline solution compared to rGO. Specifically, 0.42% Ni-NPs/rGO revealed excellent performance for glucose determination with a wide linear range (0.25 mu M to 1200 mu M), a highly reproducible response and long-term stability with the calculated detection limit of 0.01 mu M. Moreover, the reliability of the Ni-NPs/rGO sensor was confirmed by evaluating glucose concentration in human blood serum. Ab initio calculations were employed to reveal molecular interactions of glucose with Ni-NPs/rGO. The results showed Ni-13 cluster with icosahedral geometry facing the graphene sheet is the most stable structure. Furthermore, the glucose molecule was able to adsorb on the graphene sheet through Ni-13 nanoclusters and the adsorption process is closely dependent on the orientation of Ni13 nanoclusters. (C) 2017 Elsevier Ltd. All rights reserved.