In this paper, the poly-Si nanowire (NW) field-effect transistor (FET) sensor arrays were fabricated by adopting low-temperature annealing (600 °C/30 s) and feasible spacer image transfer (SIT) processes for future monolithic three-dimensional integrated circuits (3D-ICs) applications. Compared with other fabrication methods of poly-Si NW sensors, the SIT process exhibits the characteristics of highly uniform poly-Si NW arrays with well-controlled morphology (about 25 nm in width and 35 nm in length). Conventional metal silicide and implantation techniques were introduced to reduce the parasitic resistance of source and drain (SD) and improve the conductivity. Therefore, the obtained sensors exhibit >106 switching ratios and 965 mV/dec subthreshold swing (SS), which exhibits similar results compared with that of SOI Si NW sensors. However, the poly-Si NW FET sensors show the Vth shift as high as about 178 ± 1 mV/pH, which is five times larger than that of the SOI Si NW sensors. The fabricated poly-Si NW sensors with 600 °C/30 s processing temperature and good device performance provide feasibility for future monolithic three-dimensional integrated circuit (3D-IC) applications.