High electron mobility transistor (HEMT) biosensors hold great potential for realizing label-free, real-time, and direct detection. Owing to their unique properties of two-dimensional electron gas (2DEG), HEMT biosensors have the ability to amplify current changes pertinent to potential changes with the introduction of any biomolecules, making them highly surface charge sensitive. This review discusses the recent advances in the use of AlGaN/GaN and AlGaAs/GaAs HEMT as biosensors in the context of different gate architectures. We describe the fundamental mechanisms underlying their operational functions, giving insight into crucial experiments as well as the necessary analysis and validation of data. Surface functionalization and biorecognition integrated into the HEMT gate structures, including self-assembly strategies, are also presented in this review, with relevant and promising applications discussed for ultra-sensitive biosensors. Obstacles and opportunities for possible optimization are also surveyed. Conclusively, future prospects for further development and applications are discussed. This review is instructive for researchers who are new to this field as well as being informative for those who work in related fields.