A hollow sphere-nanostructured conductive polymer/metal oxide composite was synthesized and used to investigate the electrochemical behavior of glucose oxidase, demonstrating a significantly enhanced direct electron transfer ability of glucose oxidase. In particular, the long-standing puzzle of whether enzymatic glucose sensing involves an enzyme direct electron transfer process was studied. The results indicate the mechanism is indeed a glucose oxidase direct electron transfer process with competitive glucose oxidation and oxygen reduction to detect glucose. A glucose biosensor with the glucose oxidase-immobilized nanomaterial was further constructed, demonstrating superior sensitivity and reliability, and providing great potential in clinical applications.