Adhesion defects between a single crystal semiconductor layer and a support substrate are reduced to manufacture an SOI substrate achiving high bonding strength between the single crystal semiconductor layer and the support substrate. Plasma is produced by exciting a source gas, ion species contained in the plasma are added from one surface of a single crystal semiconductor substrate, and thereby forming a damage region in the single crystal semiconductor substrate; forming an insulating layer over one surface of the single crystal semiconductor substrate; a support substrate is bonded so as to face the single crystal semiconductor substrate with the insulating layer therebetween; the single crystal semiconductor substrate is heated to separate the single crystal semiconductor substrate into a single crystal semiconductor layer bonded to the support substrate and a single crystal semiconductor substrate, in the damage region; and the single crystal semiconductor layer bonded to the support substrate is pressed.