Atomic force microscopy (AFM) nanolithography was employed to fabricate poly(methyl methacrylate) (PMMA) nanochannels with a width of 80nm, a depth of 30nm, and a length of up to 8μm on a Pt-coated indium tin oxide substrate. Quasi-sigmoidal steady-state responses was observed in cyclic voltammograms for the Fe(CN)64-/Fe(CN)63- redox system in aqueous 0.1M KCl at the patterned PMMA-coated Pt surface. The PMMA nanochannels were used to prepare glucose nanobiosensors by electrodeposition of glucose oxidase in the presence of Triton X-100. The height of the resulting glucose nanobiosensor was about 80nm. The prepared glucose nanobiosensors displayed a sensitivity of 6.56AM −1 cm −2 and a response time of less than 3s. The sensitivity obtained in this study is much higher than those obtained at normal macroelectrodes. The present methodology demonstrates the feasibility of electrodeposition of glucose oxidase in PMMA nanochannels produced by AFM nanolithography for biosensing of glucose.