Glucose oxidase (GOx) was immobilized between aligned carbon nanotube films (ACNTFs) using a cross-stacking method without any purification and functionalization processes. Direct electron transfer of GOx was achieved on the ACNTFs with reversible redox peaks observed at the formal potential of –0.448 V. When applied to the determination of glucose by cyclic voltammetry, the prepared enzymatic electrode exhibited a linear range from 1 to 5 mM under mediator-free physiological conditions, which makes it a promising candidate for the development of third-generation glucose biosensors.