An SOI substrate is manufactured by a method in which a first insulating film is formed over a first substrate over which a plurality of first single crystal semiconductor films is formed; the first insulating film is planarized; heat treatment is performed on a single crystal semiconductor substrate attached to the first insulating film; a second single crystal semiconductor film is formed; a third single crystal semiconductor film is formed using the first single crystal semiconductor films and the second single crystal semiconductor films as seed layers; a fragile layer is formed by introducing ions into the third single crystal semiconductor film; a second insulating film is formed over the third single crystal semiconductor film; heat treatment is performed on a second substrate superposed on the second insulating film; and a part of the third single crystal semiconductor film is fixed to the second substrate.