Glucose oxidase can catalyze the oxidization of glucose in the mild conditions, which can be used to decorate the anode of the biofuel cells. The carbon nanotube electrode can direct contact with the flavin adenine dinucleotide cofactor of glucose oxidase by automatic absorption. Our ab initio calculations reveal that proton/electron transfer from β-D-glucose to isoaloxazine ring occurs via a proton-coupled double-electron transfer mechanism according to a model including deprotonated glucose and lumiflavin with a high energy barrier of 30.5 kcal/mol. The similar result is obtained from the examination of the corresponding reaction in glucose oxidase at the ONIOM(M06–2X/6-31 + G(d,p):amber) level. When the isoalloxazine ring directly interacts with the surface of carbon nanotube in the glucose-oxidase-modified electrode, however, the reaction occurs through a π-π-driven proton-coupled double-electron transfer mechanism with a low energy barrier (0.2–12.0 kcal/mol). This is because that the π-π interaction plays a central role in promoting electron transfer from substrate to flavin. These findings indicate that the association of glucose oxidase and carbon nanotube is a potential application for the effective electrode of enzymatic biofuel cell.