In this paper, we have developed a biofriendly and high sensitive apo-GOx (inactive form of glucose oxidase)-modified gold nanoprobe for quantitative analysis of glucose and imaging of glucose consumption in living cells. This detection system is based on fluorescence resonance energy transfer between apo-GOx modified AuNPs (Au nanoparticles) and dextran-FITC (dextran labeled with fluorescein isothiocyanate). Once glucose is present, quenched fluorescence of FITC recovers due to the higher affinity of apo-GOx for glucose over dextran. The nanoprobe shows excellent selectivity toward glucose over other monosaccharides and most biological species present in living cells. A detection limit as low as 5 nM demonstrates the high sensitivity of the nanoprobe. Introduction of apo-GOx, instead of GOx, can avoid the consumption of O2 and production of H2O2 during the interaction with glucose, which may exert effects on normal physiological events in living cells and even lead to cellular damage. Due to the low toxicity of this detection system and reliable cellular uptake ability of AuNPs, imaging of intracellular glucose consumption was successfully realized in cancer cells.