It is an object of the present invention is to provide a method of manufacturing an SOI substrate provided with a single-crystal semiconductor layer which can be practically used even when a substrate having a low heat-resistant temperature, such as a glass substrate or the like, is used, and further, to manufacture a semiconductor device with high reliability by using such an SOI substrate. A semiconductor layer which is separated from a semiconductor substrate and bonded to a supporting substrate having an insulating surface is irradiated with electromagnetic waves, and the surface of the semiconductor layer is subjected to polishing treatment. At least part of a region of the semiconductor layer is melted by irradiation with electromagnetic waves, and a crystal defect in the semiconductor layer can be reduced. Further, the surface of the semiconductor layer can be polished and planarized by polishing treatment.