Abstract: High electron mobility transistor (HEMT) biosensors based on III–V semiconductor materials, leveraging the high mobility and near-surface charge sensitivity of the two-dimensional electron gas (2DEG) channel, offer unique advantages in charge transduction and signal amplification for label-free biodetection. However, insufficient long-term device stability and limited biointerface recognition reliability remain major obstacles to their practical applications. This review starts from HEMT device physics, summarizing gate structure engineering strategies for suppressing signal drift; then focuses on the rational design of sensing interfaces, discussing technological approaches to overcome Debye shielding and enhance charge coupling efficiency; and finally reviews recent progress in applying HEMT biosensors across clinical diagnostics, health monitoring, and environmental detection. Looking forward, with the deeper integration of rational interface design and device structural engineering, HEMT biosensors are expected to become a core technology platform for next-generation point-of-care testing (POCT) and home-based health monitoring.