A manufacturing method of a glass substrate with a hole having a diameter ϕf, and with a thickness θf includes setting a thickness θ1 of a glass plate that is to be processed; preparing a glass plate with the thickness θ1, having first and second surfaces opposite to each other; forming one initial characteristic object or two or more initial characteristic objects in the glass plate by irradiating the glass plate with a laser from a side of the first surface of the glass plate, the initial characteristic object having a size of a diameter ϕ1 on the first surface; and performing wet etching for the glass plate having the initial characteristic object, so that from the initial characteristic object a hole having the diameter ϕf on the first surface is formed, and a thickness of the glass plate is adjusted from θ1 to a target value of θf.