A single crystal semiconductor layer is formed over a substrate having an insulating surface by the following steps: forming an ion doped layer at a given depth from a surface of a single crystal semiconductor substrate; performing plasma treatment to the surface of the single crystal semiconductor substrate; forming an insulating layer on the single crystal semiconductor substrate to which the plasma treatment is performed; bonding the single crystal semiconductor substrate to the substrate having the insulating surface with an insulating layer interposed therebetween; and separating the single crystal semiconductor substrate using the ion doped layer as a separation surface. As a result, a semiconductor substrate in which a defect in an interface between the single crystal semiconductor layer and the insulating layer is reduced can be provided.