We fabricated a non-enzymatic Ni–Au alloy nanowire electrochemical glucose sensor on a p-silicon-based anodic aluminum oxide (AAO) template. The advantages of the Ni–Au glucose sensor are its high sensitivity and stability, and fast reaction. In contrast to a traditional enzymatic glucose sensor, the non-enzymatic glucose sensor is reusable, enabling its long-term use by patients with diabetes. Although the reduction voltages of Ni and Au are considerably different, the Ni–Au alloy nanowires used in the sensor were fabricated successfully via the adjustment of the electrodeposition parameters. The Ni–Au alloy nanowires exhibited a high crystallinity and a uniform arrangement with an average height of 750 nm. The Ni–Au alloy glucose sensor exhibited a linear range of 0–3 mM, a sensitivity of 1893 μA/mMcm2, and a detection limit of 1 μM. Superior selectivity and stability over at least 30 days were also observed. The characteristics show that Ni–Au alloy nanowires have excellent glucose-sensing performance compared with single noble metal nanowires. The silicon-based AAO template also serves as a strong mechanical support of the Ni–Au nanowires used for sensing. To our knowledge, our fabricated sensor is the first non-enzymatic glucose sensor to use Ni–Au alloy nanowires.