In order to enhance the performance of AlGaN/GaN high electron mobility transistor (HEMT) biosensor, millimeter grade AlGaN/GaN HEMT structure have been designed and successfully fabricated. Factors influencing the capability of the AlGaN/GaN HEMT biosensor are analyzed. UV/ozone is used to oxidize GaN surface and then 3-aminopropyl trimethoxysilane (APTES) self-assembled monolayer can be bound to the sensing region. This serves as a binding layer in the attachment of prostate specific antibody (anti-PSA) for prostate specific antigen detection. The millimeter grade biomolecule-gated GaN/AlGaN HEMT sensor shows a quick response when the target prostate specific antigen in a buffer solution is added to the antibody-immobilized sensing area. The detection capability of this biomolecule-gate sensor estimated to be below 0.1 pg/ml level using a 21.5 mm2 sensing area, which is the best result of GaN/AlGaN HEMT biosensor for PSA detection till now. The electrical result of the biomolecule-gated GaN/AlGaN HEMT biosensor suggests that this biosensor might be a useful tool for the prostate cancer screening.