In this article, silicon on insulator (SOI) was fabricated by separation by implanted oxygen (SIMOX) layer transfer process. The thickness uniformity of the top Si (superficial Si of the SOI) evaluated by spectroscopic ellipsometry remains constant through the etch-stop thinning process. A defect-free top Si was observed by transmission electron microscopy, as well as its atomic-scale sharp interface that was adjacent to the buried oxide (BOX) layer. The top Si/BOX interface properties, including upper BOX surface roughness and interface state density, were investigated by atomic force microscopy (AFM) and pseudo-metal-oxide-semiconductor method, respectively. In addition, the surface morphology of the square pit and square hole defect of the top Si were observed. Using AFM, it is found that the morphology depends on the top Si/BOX interface morphology of the donor wafer and can be improved by optimizing the annealing process. The square hole defect caused by anisotropic etching is discussed in detail, and it is proven to originate from the defects in the SIMOX BOX.