A manufacturing method of an SOI substrate and a manufacturing method of a semiconductor device are provided. When a large-area single crystalline semiconductor film is formed over an enlarged substrate having an insulating surface, e.g., a glass substrate by an SOI technique, the large-area single crystalline semiconductor film is formed without any gap between plural single crystalline semiconductor films, even when plural silicon wafers are used. An aspect of the manufacturing method includes the steps of disposing a first seed substrate over a fixing substrate; tightly arranging a plurality of single crystalline semiconductor substrates over the first seed substrate to form a second seed substrate; forming a large-area continuous single crystalline semiconductor film by an ion implantation separation method and an epitaxial growth method; forming a large-area single crystalline semiconductor film without any gap over a large glass substrate by an ion implantation separation method again.