This paper demonstrates an AlGaN/GaN ion-sensitive heterostructure field-effect transistors (ISHFETs)-based pH sensors with an aluminum oxide (Al2O3) to serve as a passivation layer and a sensing membrane at the same time. Al2O3 was deposited by the ultrasonic spray pyrolysis deposition (USPD) method. It was found that the ISHFET with Al2O3 showed a higher pH sensitivity of 55.6 mV/pH, a faster response speed of 3 s (from pH 4 to pH 7) and 7 s (from pH 7 to pH 10), a lower hysteresis voltage of 4.3 mV, and a smaller drift rate of 1.25 mV/h (at pH 7) compared with the ISHFET without passivation. In addition, the ISHFET with an Al2O3 passivation layer exhibited significant surface potential variation and low degradation rate. These results suggest that the performance of ISHFET improved using USPD-grown Al2O3.