A glassy carbon electrode was modified with β-manganese dioxide (β-MnO2), and glucose oxidase (GOx) was immobilized on its surface. The β-MnO2 nanowires were prepared by a hydrothermal method and characterized by scanning electron microscopy and powder X-ray diffraction. They were then dispersed in Nafion solution and cast on the glassy carbon electrode (GCE) to form an electrode modified with β-MnO2 nanowires that exhibits improved sensitivity toward hydrogen peroxide. If GOx is immobilized in the surface, the β-MnO2 acts as a mediator, and Nafion as a polymer backbone. The fabrication process was characterized by electrochemical impedance spectroscopy, and the sensor and its materials were characterized by cyclic voltammetry and amperometry. The biosensor enables amperometric detection of glucose with a sensitivity of 38.2 μA · mM−1 · cm−2, and a response time of < 5 s. This study also demonstrates the feasibility of realizing inexpensive, reliable, and high-performance biosensors using MnO2 nanowires.