Nanowire field-effect transistors (NW-FETs) are emerging as powerful sensors for detection of chemical/biological species with various attractive features including high sensitivity and direct electrical readout. Yet to date there have been limited systematic studies addressing how the fundamental factors of devices affect their sensitivity. Here we demonstrate that the sensitivity of NW-FET sensors can be exponentially enhanced in the subthreshold regime where the gating effect of molecules bound on a surface is the most effective due to the reduced screening of carriers in NWs. This principle is exemplified in both pH and protein sensing experiments where the operational mode of NW-FET biosensors was tuned by electrolyte gating. The lowest charge detectable by NW-FET sensors working under different operational modes is also estimated. Our work shows that optimization of NW-FET structure and operating conditions can provide significant enhancement and fundamental understanding for the sensitivity limits of NW-FET sensors.