It is an object of the present invention to obtain a large-sized SOI substrate by providing a single-crystal silicon layer over a large-sized glass substrate in a large area. After a plurality of rectangular single-crystal semiconductor substrates each provided with a separation layer are aligned over a dummy substrate and both of the substrates are fixed with a low-temperature coagulant, the plurality of single-crystal semiconductor substrates are bonded to a support substrate; the temperature is raised up to a temperature, at which the low-temperature coagulant does not to have a bonding effect, so as to isolate the dummy substrate and the single-crystal semiconductor substrates; heat treatment is performed to separate part of the single-crystal semiconductor substrates, along a boundary of the respective separation layers; and single-crystal semiconductor layers are provided over the support substrate.