A novel monocrystalline AlN interfacial layer formation method is proposed to improve the device performance of the fully recessed-gate Al2O3/AlN/GaN Metal-Insulator-Semiconductor High Electron Mobility Transistors (MIS-HEMTs), which is achieved by plasma-enhanced atomic layer deposition (PEALD) and in situ N2plasma annealing (NPA). Compared with the traditional RTA method, the NPA process not only avoids the device damage caused by high temperatures but also obtains a high-quality AlN monocrystalline film that avoids natural oxidation by in situ growth. As a contrast with the conventional PELAD amorphous AlN,C-Vresults indicated a significantly lower interface density of states (Dit) in a MISC-Vcharacterization, which could be attributed to the polarization effect induced by the AlN crystal from the X-ray Diffraction (XRD) and Transmission Electron Microscope (TEM) characterizations. The proposed method could reduce the subthreshold swing, and the Al2O3/AlN/GaN MIS-HEMTs were significantly enhanced with ~38% lower on-resistance atVg= 10 V. What is more, in situ NPA provides a more stable threshold voltage (Vth) after a long gate stress time, and ΔVthis inhibited by about 40 mV underVg,stress= 10 V for 1000 s, showing great potential for improving Al2O3/AlN/GaN MIS-HEMT gate reliability.