In this study, we developed polycrystalline silicon (poly-Si) nanowires by a sidewall spacer technique and employed them to fabricate nanowire field-effect transistor (NWFET) devices for biomedical sensing applications. The impact of post-fabrication thermal treatments on the electrical performance of the poly-Si NWFET biosensors was systematically investigated. Among the tested conditions, the devices treated at 400 °C in vacuum exhibited significantly enhanced electrical characteristics. These optimized biosensors were then used for solution pH detection, demonstrating high sensitivity and a clear linear correlation between drain current and pH. Furthermore, real-time pH sensing experiments confirmed the biosensors’ capability to respond rapidly and accurately to changes in pH, highlighting both their sensitivity and response speed. Overall, the developed poly-Si NWFET biosensors show strong potential for advancing medical diagnostics by enabling early disease detection and supporting personalized healthcare strategies.