A manufacturing method of a semiconductor substrate is provided, in which a bonding strength can be increased even when a substrate having low heat resistant temperature, e.g., a glass substrate, is used. Heat treatment is conducted at a temperature higher than or equal to a strain point of a support substrate in an oxidation atmosphere containing halogen, so that a surface of a semiconductor substrate is covered with an insulating film. A separation layer is formed in the semiconductor substrate. A blocking layer is provided. Then, heat treatment is conducted in a state in which the semiconductor substrate and the support substrate are superposed with the silicon oxide film therebetween, at a temperature lower than or equal to the support substrate, so that a part of the semiconductor substrate is separated at the separation layer. In this manner, a single crystal semiconductor layer is formed on the support substrate.