<p>A highly sensitive and stable biosensor was crucial for the early screening and treatment of diseases. As one of the most promising and powerful platforms, AlGaN/GaN HEMT biosensors have enormous potential owing to their distinctive 2DEG channel features. While gold nanomaterials offer great potential for creating highly sensitive sensing surfaces due to their nanoscale size, their lack of robustness and inconsistent concentration on the HEMT surface hinders the reliability and reproducibility of biosensors. Herein, gold nanoislands (AuNis) are utilized for the first time as a sensing membrane on AlGaN/GaN HEMT devices for biosensing applications. The high sensitivity for pH detection verified the excellent AuNis sensing capability. For biosensing applications, glutathione S-transferase–p21-activated kinase1–GTPase-binding domain (GST-PAK1-GBD; residues 56–272) served as a bioreceptor for small Rho GTPases in Jurkat T-cell lysate detection, achieving high current and voltage sensitivities of 9.10% and 33.00% at 3 × 10⁻⁷&#xa0;g/mL, respectively. Benefitting from the large surface binding site of the AuNis sensing surface, a wider range of Jurkat T-cell lysate detection from 3 × 10⁻¹⁶ to 3 × 10⁻⁷&#xa0;g/mL and an ultra-low limit of detection of 3 × 10⁻¹⁶&#xa0;g/mL are obtained. Furthermore, excellent reproducibility with a reported correlation coefficient (R² ≥ 0.950) across multiple sensors and &gt; 98% signal recovery after regeneration cycles further validates the sensor’s performance and reusability. The modification of the sensing surface with AuNis marks a significant advancement toward the development of robust and reproducible biosensors.</p>