Human epidermal growth factor receptor-3 (HER-3) plays a key role in the growth and metastasis of cancer cells. The detection of HER-3 is very important for early screening and treatment of cancer. The AlGaN/GaN-based ion-sensitive heterostructure field effect transistor (ISHFET) is sensitive to surface charges. This makes it a promising candidate for the detection of HER-3. In this paper, we developed a biosensor for the detection of HER-3 with AlGaN/GaN-based ISHFET. The AlGaN/GaN-based ISHFET biosensor exhibits a sensitivity of 0.53 ± 0.04 mA/dec in 0.01 M phosphate buffer saline (1× PBS) (pH = 7.4) solution with 4% bovine serum albumin (BSA) at a source and drain voltage of 2 V. The detection limit is 2 ng/mL. A higher sensitivity (2.20 ± 0.15 mA/dec) can be achieved in 1× PBS buffer solution at a source and drain voltage of 2 V. The AlGaN/GaN-based ISHFET biosensor can be used for micro-liter (5 μL) solution measurements and the measurement can be performed after incubation of 5 min.