An apparatus and method to inspect a defect of a substrate. Since a recess of an under layer of a substrate is darker than a projection of a top layer, a ratio of a value of a secondary electron signal (of an SEM) of the under layer to a value of the top layer may be increased to improve a pattern image used to inspect an under layer defect. Several conditions under which electron beams are irradiated may be set, and the pattern may be scanned under such conditions. Secondary electron signals may be generated according to the conditions and converted into image data to display various pattern images. Scan information on the images may be stored with positional information on the substrate. Each of scan information on the pattern images may be calculated to generate a new integrated image.