AbstractSensing technology for biomolecules has played a key role for maintaining a high quality of life. Various kinds of sensing techniques have been developed for detecting biomolecules. Among the detection techniques which have been reported so far, non‐enzymatic glucose detection, based on electrochemical mechanisms, has attracted great interest as a facile route for monitoring glucose levels in the blood. In this method, designing nanostructured electrodes is a promising strategy to improve detection performance. Owing to their fascinating physicochemical properties, including superior electrical and electrocatalytic properties, mesoporous metal films offer a wide range of useful applications which cannot be achieved by other mesoporous materials. In this work, mesoporous Au films (MpGFs) are synthesized by an effective and simple electrochemical process in the presence of a surfactant, where the mesoporous structure can be controlled by the size of the polymeric micelles. The micelles are removed using a tetrahydrofuran‐based solvent‐extraction method. The activity of MpGFs towards glucose oxidation is evaluated under a fixed potential in alkaline solution. Under optimal potentials, the current response for other interfering molecules is minimal, while the current response for glucose is maximal. A linear relationship between the current response and the glucose content is observed over a wide range of concentrations. Thus, our MpGF shows an excellent performance for electrochemical glucose sensing applications.