Protein conformation associates with particular properties of proteins and relates to protein-mediated diseases. Detailed elucidation of secondary and tertiary formation, stability, and the structural and dynamic properties of proteins has been one of the main topics studied in chemistry and biology. In this work, the conformation changes in glucose oxidase (GOx) induced by the graphene oxide (GO) sheets were studied in detail by various spectroscopic techniques including ultraviolet–visible (UV–vis) absorption, fluorescence, and circular dichroism (CD) spectroscopy. The results indicated that GOx underwent substantial conformation changes after assembling on the surface of GO. The interaction of GOx with GO could induce the exposure of the FAD (flavin adenine dinucleotide) moiety to solvent and transfer tryptophan (Trp) residues to a more hydrophobic environment. The calculation from CD spectra showed that GO could induce the conversion of α-helix to β-sheet structures, even unfolding of the protein. These alterations in the conformation of GOx resulted in a significant decrease in the catalytic activity of the enzyme in glucose oxidation. The possible reasons for these conformation changes in GOx are also discussed. This work not only provides insight into the interaction between atomically flat graphitic structures and various biological systems but also creates a framework for analyzing the biosafety of nanomaterials in terms of the biological behavior of biomacromolecules. The results obtained here can direct the further applications of the nanomaterials.