High‐performance electrodes for in vivo electrochemical detection of glucose (GO) are highly desirable. In this work, we propose a new approach to efficiently and precisely prepare a Au nanowire array electrode (ANAE) with a line width of 78 nm and a large sensor area of 60 mm×60 mm for GO detection. In this approach three techniques, i.e., vacuum sputtering‐deposition, holography photolithography, and argon ion‐beam etching, are integrated. The fabricated ANAE exhibits good performance for GO detection. A linear amperometric response to the oxidation of GO in a concentration range of 0.4–10 mM is observed. The ANAE is characterized by its high detection sensitivity, selectivity, stability and good biocompatibility. All of these make it a promising tool for GO detection and other relevant applications.