The radiation response of Al2O3 on silicon substrate under gamma-rays is studied in this article. The atomic layer deposited Al2O3 based metal-oxide-semiconductor structures were irradiated under gamma-ray with the total dose of 1.2 Mrad(Si)/2.5 Mrad(Si)/4 Mrad(Si). The generation, transportation and trapping characteristics of radiation induced charges were studied by using electronic, physical and chemical methods. Firstly, the radiation induced trapped charge density in Al2O3 is up to 1012 cm−2, with the effective trapping efficiency of 7–20% under irradiation. Secondly, the leakage current through Al2O3 changes little with the increase of radiation total dose. Thirdly, oxygen vacancy in Al2O3 and O dangling bonds and Al-Si metallic bonds at Al2O3/Si interface are dominant radiation induced defects in Al2O3/Si system, and the valence band offset between Al2O3 and Si is found to decrease after irradiation. From the results we can see that Al2O3 is radiation resistant from the aspect of leakage current and crystallization characteristics, but the radiation induced charge trapping and new defects in Al2O3/Si structure cannot be ignored. This paper provides a reference for the space application of Al2O3 based MOS devices.