Using a silicon nanowire field-effect transistor (SiNW-FET) for biomolecule detections, we selected 3-(mercaptopropyl)trimethoxysilane (MPTMS), N-[6-(biotinamido)hexyl]-3(')-(2(')-pyridyldithio) propionamide (biotin-HPDP), and avidin, respectively, as the designated linker, receptor, and target molecules as a study model, where the biotin molecules were modified on the SiNW-FET to act as a receptor for avidin. We applied high-resolution scanning Kelvin probe force microscopy (KPFM) to detect the modified/bound biomolecules by measuring the induced change of the surface potential (ΔΦ(s)) on the SiNW-FET under ambient conditions. After biotin-immobilization and avidin-binding, the ΔΦ(s) on the SiNW-FET characterized by KPFM was demonstrated to correlate to the conductance change inside the SiNW-FET acquired in aqueous solution. The ΔΦ(s) values on the SiNW-FET caused by the same biotin-immobilization and avidin-binding were also measured from drain current versus gate voltage curves (I(d)-V(g)) in both aqueous condition and dried state. For comparison, we also study the ΔΦ(s) values on a Si wafer caused by the same biotin-immobilization and avidin-binding through KPFM and ζ potential measurements. This study has demonstrated that the surface potential measurement on a SiNW-FET by KPFM can be applied as a diagnostic tool that complements the electrical detection with a SiNW-FET sensor. Although the KPFM experiments were carried out under ambient conditions, the measured surface properties of a SiNW-FET are qualitatively valid compared with those obtained by other biosensory techniques performed in liquid environment.