Yttria-stabilized zirconia (YSZ) thermal barrier coatings (TBCs) are prepared by plasma spraying. Thermally grown oxide (TGO) would be formed between YSZ topcoat and bond coat after 50 h thermal service for YSZ TBCs. The electron back scattered diffraction (EBSD) results reveal that the TGO layer is composed of alpha-Al2O3 and cubic Al2NiO4 layers. Measured values of TGO thickness from the 2D-SEM image are greater than or equal to its real thickness due to the fact that the TGO layer is much rolling so that up and down surfaces of the TGO can't be completely perpendicular to the cross-section direction confirmed by 3D-SEM. Furthermore, 3D-SEM results reveal that the real thickness of TGO layer is 3.10 mu m instead of 7.1 mu m. In addition, 3D-EBSD confirmed that alpha-Al2O3 layer in TGO is composed of single layer of grains and Al2NiO4 layer consist of multilayer of grains while alpha-Al2O3 layer is mixed with single layer and multilayer of alpha-Al2O3 grains from observation of the 2D-EBSD image. It provides a new method to characterize real thickness and microstructure of TGO, which is also applied to other film materials. (C) 2016 Elsevier Inc. All rights reserved.