An SOI substrate is manufactured by the following steps: a semiconductor substrate is irradiated with an ion beam in which the proportion of H2O+ to hydrogen ions (H3+) is lower than or equal to 3%, preferably lower than or equal to 0.3%, whereby an embrittled region is formed in the semiconductor substrate; a surface of the semiconductor substrate and a surface of a base substrate are disposed so as to be in contact with each other, whereby the semiconductor substrate and the base substrate are bonded; and a semiconductor layer is separated along the embrittled region from the semiconductor substrate which is bonded to the base substrate by heating the semiconductor substrate and the base substrate, so that the semiconductor layer is formed over the base substrate.