A defect detection apparatus (1) comprises an image pickup part (3) for picking up an image of a substrate (9) to acquire a grayscale target image, from which pixel values of the target image are sequentially outputted to a defect detector (43). The defect detector (43) compares the target image with a reference image to generate a defect region image representing regions of defects included in a plurality of predetermined inspection regions, to be stored in a defect region image memory (44). A computer (5) obtains an area and a barycentric position of each of the defects in the defect region image to specify an inspection region including the defect and performs limitation (i.e., selection) of defects on the basis of a defect detection condition set for each inspection region on the area of defect. It is possible to detect defects with high efficiency by using a defect detection condition on a different area of defect for each inspection region.