An SOI substrate and a manufacturing method of the SOI substrate, by which enlargement of the substrate is possible and its productivity can be increased, are provided. A step (A) of cutting a first single crystal silicon substrate to form a second single crystal silicon substrate which has a chip size; a step (B) of forming an insulating layer on one surface of the second single crystal silicon substrate, and forming an embrittlement layer in the second single crystal substrate; and a step (C) of bonding a substrate having an insulating surface and the second single crystal silicon substrate with the insulating layer therebetween, and conducting heat treatment to separate the second single crystal silicon substrate along the embrittlement layer, and forming a single crystal silicon thin film on the substrate having an insulating surface, are conducted.