A method of manufacturing a multi-layered semiconductor substrate comprising:a step of forming a first insulation film on the main surface of a semiconductor substrate composed of single crystals,a step of forming a first linear opening of a predetermined size reaching the semiconductor substrate at a predetermined position of the first insulation film,a step of forming second opening with the opening area of 25 .mu.m.sup.2 or less and reaching the semiconductor substrate along the first opening to the first insulation film at a position a spaced apart at least by 10 .mu.m from the outer edge of the first opening,a step of forming a semiconductor layer composed of non-single crystals on the first insulation film also including the inside of the first and the second openings,a step of forming a second insulation film on the semiconductor layer,a step of supplying heat energy to the semiconductor layer by scanning in the direction from the first opening toward the second opening, and melting the semiconductor layer with the heat energy thereby single-crystallizing the semiconductor layer, anda step of removing the second insulation film.