Low operating voltages, rapid response, and high-throughput fabrication compatibility are key advantages for the development of electrolyte-gated field effect transistors (EGFETs) for biological sensing. Among the key components in EGFET biosensors, electrolyte materials are relatively less investigated, especially alternatives to water-based liquid electrolytes such as ionic liquids, ion gels, polyelectrolytes, and solid polymer electrolytes. These electrolytes enable portable devices and environmental stability superior to their water-based liquid alternatives. In this review, we offer an up-to-date evaluation of the state of EGFET research and gauge the strengths and limitations of high-performance electrolytes for use in EGFET biosensor applications as well as the potential for computer-aided design of such sensing platforms. The recent progress of EGFET biosensors for some popular analytes are reviewed and the performance of these alternative electrolytes in transistor biosensing is assessed. The challenges and opportunities for electrolytes in EGFETs are discussed for future research directions in this field.