We investigated the characteristics of Al2O3 gate pH-ISFET which was fabricated by using the standard complementary metal oxide semiconductor (CMOS)-process techniques. The Al2O3 film used to sensing membrane was deposited by atomic layer deposition (ALD). Then, thermal temperature annealing process of the Al2O3 film was performed in O2 ambient for 40 min at a temperature of 500°C, 600°C, 800°C and 900°C. We measured the I D– V D, I D– V G and transconductance of fabricated FET device in order to confirm stability of device before the fabrication of pH-ISFET. Then, the package process was performed. To investigate the pH response characteristics, we measured the I–V curves of the Al2O3 gate pH-ISFET sensor using a 4155 probe station. Form the measured results, we confirmed that sensitivity, hysteresis, and long-term stability of the Al2O3 pH-ISFET showed the changed characteristics at various annealing temperature. The characteristics of Al2O3 gate pH-ISFET annealed at 600°C indicated the best results of the high sensitivity (56 mV/pH), low hysteresis (0.5 ~ 0.7 mV), and low drift (1.35 mV/h) in comparison to Al2O3 gate pH-ISFETs annealed other conditions. As measured results, we confirmed that sensitivity, hysteresis, and long-term stability of Al2O3 gate pH-ISFET depend on the thermal annealing temperature of Al2O3 film and also it is the very important parameter in the pH-ISFET.