Protein conformational changes may be associated with particular properties such as its function, transportation, assembly, tendency to aggregate, and potential cytotoxicity. In this study, the mechanism of the effects of thermal unfolding of proteins on their catalytic activities and conformational structures were studied by utilizing glucose oxidase (GOx) as a model protein. The characteristic kinetic constants for the enzymatic reaction were evaluated by the use of an electrochemical approach under a substrate-saturated condition. A combination of quantitative second-derivative infrared analysis, two-dimensional infrared correlation spectroscopy (2D IR), and theoretical calculation was used to elucidate the conformational structures that were responsible for inactivation and denaturation of GOx induced by heat. The IR analysis demonstrated that the conformational structures of GOx, especially the a-helix and unordered structures, were greatly dependent on the system temperature. Thermal treatment resulted in the increase of the unordered structure accompanied by the loss of the a-helical structure in GOx conformation. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations revealed that thermal treatment could significantly alter the electronic characteristics and the intramolecular electron transfer ability of FAD (flavin adenine dinucleotide), and hydrogen bond networks formed between FAD and the amino acid residues around the cofactor, leading to the change of the secondary structure and the catalytic activity of GOx. The study essentially paves an effective approach to investigation of the mechanism of protein unfolding.