Zinc oxide tetrapods (ZnO-Ts) were synthesized by flame transport synthesis using Zn microparticles. This work herein reports a systematical study on the structural, optical and electrochemical properties of the ZnO-Ts. The morphology of the ZnO-Ts was confirmed by scanning electron microscopy (SEM) as joint structures of four nano-microstructured legs, of which the diameter of each leg is 0.7-2.2 μm in average from the tip to the stem. The ZnO-Ts were dispersed in glucose solution to study the luminescence as well as photocatalytic activity in a mimicked biological environment. The photoluminescence (PL) intensity in the ultraviolet (UV) region quenches with linear dependence to increased glucose concentration up to 4 mM. The ZnO-Ts were also attached with glucose oxidase (GOx) and over coated with a thin film of Nafion to form active layers for electrochemical glucose sensing. The attachment of GOx and coating of Nafion were confirmed by infrared spectroscopy (FT-IR). Furthermore, the current response of the active layers based on ZnO-Ts was investigated by cyclic voltammetry (CV) in various glucose concentrations. Stable current response of glucose was detected with linear dependence to glucose concentration up to 12 mM, which confirms the potential of ZnO-Ts for biomolecule sensing applications.