This paper uses H2O2 oxidation technique to grow Al2O3 on AlGaN/GaN heterostructure. The H2O2-grown-Al2O3 is served as a sensing membrane and a surface passivation layer. The contact angle of the ion-sensitive field-effect transistor (ISFET) with the H2O2-grown-Al2O3 is improved from 66.5° to 40.6° and this phenomenon indicates that the hydrophile characteristic is improved after the H2O2 treatment. The drain-source current ( $I _{\textrm {DS}}$ ) is improved $\sim 32$ % after the H2O2 oxidation due to the passivation effect. In addition, extrinsic transconductance ( $\mathrm{g}_{m}$ ) characteristics of the transistors are investigated. The pH sensitivity is also improved from 41.6 to 55.2 mV/pH for the ISFET with H2O2-grown-Al2O3. Furthermore, the ISFET with the H2O2 treatment exhibits better transient characteristics compared with the ISFET without the H2O2 treatment. The sensitivity parameter ( $\beta $ ) and the relationship between surface potential ( $\psi _{s}$ ) and pH value are investigated by theoretical calculation. The hysteresis phenomenon and the drift effect can be improved using the present H2O2-grown Al2O3 as the sensing membrane and the passivation layer.