This study fabricated a multiple poly-Si nanowires sensor through a top-down method and immobilized glucose oxidase on the multiple nanowires for determining glucose concentration. The proposed sensor is 340 nm in width and uses five physically identical and parallel nanowires. The sensor contained nanowires of various lengths (3, 5, and 10 µm). Experimental results showed that sensor sensitivity is inversely proportional to nanowire length. The sensor with 3 µm in nanowire length exhibited a theoretical resolution of 0.003 mg/dL and the highest sensitivity of 0.03 µA/(mg/dL). Furthermore, the proposed sensor retains this performance when reused for up to 10 applications.