Highly oriented single-crystal ZnO nanotube (ZNT) arrays were prepared by a two-step electrochemical/chemical process on indium-doped tin oxide (ITO) coated glass in an aqueous solution. The prepared ZNT arrays were further used as a working electrode to fabricate an enzyme-based glucose biosensor through immobilizing glucose oxidase in conjunction with a Nafion coating. The present ZNT arrays-based biosensor exhibits high sensitivity of 30.85 μA cm−2 mM−1 at an applied potential of +0.8 V vs. SCE, wide linear calibration ranges from 10 μM to 4.2 mM, and a low limit of detection (LOD) at 10 μM (measured) for sensing of glucose. The apparent Michaelis−Menten constant KMapp was calculated to be 2.59 mM, indicating a higher bioactivity for the biosensor.