In a defect detection apparatus 1, in a reference image inspection circuit 42 compared are a reference image representing a pattern in a die which is determined as a reference on a substrate 9 and a plurality of supervisory images which represent patterns in selected block areas, respectively, to detect defects included in the reference image. Subsequently, in the target image inspection circuit 44, a target image representing a pattern in another die and the reference image are compared to detect a plurality of defect candidates included in the target image. Then, a defect detector 45 excludes defect candidates overlapping with the defects included in the reference image from the plurality of defect candidates on the basis of at least positional information of the defects included in the reference image. This makes it possible to detect defects existing in the pattern in another die accurately while eliminating effects of the defects existing in the pattern in the die which is determined as the reference.