Herein, we propose a transparent high-performance extended-gate ion-sensitive field-effect transistor (EG-ISFET) using an electrospun indium-tin-oxide (ITO) nanofiber sensing membrane with a high specific surface area. Electrospinning is a simple and effective technique for forming nanofibers. Nevertheless, one-step calcination, such as conventional thermal annealing or microwave annealing, cannot sufficiently eliminate the inherent defects of nanofibers. In this study, we efficiently removed residual polymers and internal impurities from nanofibers via a two-step calcination process involving combustion and microwave annealing. Moreover, Ar plasma treatment was performed to improve the electrical characteristics of ITO nanofibers. Conformally coated thin-film sensing membranes were prepared as a comparative group and subjected to the same calcination conditions to verify the effect of the nanofiber sensing membrane. The characteristics of the ITO nanofiber and ITO thin-film sensing membranes were evaluated using scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), optical transmittance, and conductivity. Moreover, the sensor operation of the EG-ISFETs is evaluated in terms of sensitivity and non-ideal behaviors. The optimized process improves the sensor characteristics and sensing membrane quality. Therefore, the ITO nanofiber sensing membrane improves the sensitivity and stability of the EG-ISFET, suggesting its applicability as a high-performance biochemical sensor.