In this research, we used a polycrystalline silicon nanowire field-effect transistor (poly-Si NWFET) as a biosensor that employs the sidewall spacer technique instead of an expensive electron beam lithography method. When compared with commercial semiconductor processes, the sidewall spacer technique has the advantages of simplicity and low cost. In this study, we employed a novel poly-Si NWFET device for real-time, label-free, and ultrahigh-sensitivity detection of prostate-specific antigen (PSA) in human serum. Since serum proteome is very complex containing high levels of salts and other interfering compounds, we hereby developed a standard operating procedure for real-sample pretreatment to keep a proper pH value and ionic strength of the desalted serum and also utilized Tween 20 to serve as the passivation agent by surface modification on the NWFET to reduce nonspecific binding for medical diagnostic applications. We first modified 3-aminopropyltriethoxysilane on the surface of a poly-Si nanowire device followed by glutaraldehyde functionalization, and the PSA antibodies were immobilized on the aldehyde terminal. While PSA was prepared in the buffers to maintain an appropriate pH value and ionic strength, the results indicated that the sensor could detect trace PSA at less than 5 fg/mL in a microfluidic channel. The novel poly-Si NWFET is developed as a diagnostic platform for monitoring prostate cancer and predicting the risk of early biochemical relapse.