Interface characterization was carried out on Al2O3/GaN structures using epitaxial n-GaN layers grown on free-standing GaN substrates with relatively low dislocation density (<3 × 106 cm−2). The Al2O3 layer was prepared by atomic layer deposition. The as-deposited metal-oxide-semiconductor (MOS) sample showed a significant frequency dispersion and a bump-like feature in capacitance-voltage (C–V) curves at reverse bias, showing high-density interface states in the range of 1012 cm−1 eV−1. On the other hand, excellent C–V characteristics with negligible frequency dispersion were observed from the MOS sample after annealing under a reverse bias at 300 °C in air for 3 h. The reverse-bias-annealed sample showed state densities less than 1 × 1011 cm−1 eV−1 and small shifts of flat-band voltage. In addition, the C–V curve measured at 200 °C remained essentially similar compared with the room-temperature C–V curves. These results indicate that the present process realizes a stable Al2O3/GaN interface with low interface state densities. This work was partially supported by JSPS KAKENHI Grant No. JP16H06421 and Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Next-generation power electronics” (Funding Agency: NEDO).