In a defect detection apparatus, images of first to third inspection areas on a substrate are picked up to acquire first to third images. A positional difference acquisition part (51) acquires a first difference vector between the first image and the second image and a second difference vector between the second image and the third image. A differential image generation part (52) generates a first differential image between the first image and the second image while adjusting a position of the first image to the second image on the basis of the first difference vector and a second differential image between the second image and the third image while adjusting a position of the second image to the third image on the basis of the second difference vector. Then, a position of the second differential image is adjusted to the first differential image on the basis of the second difference vector and the second differential image after position adjustment and the first differential image are compared with each other, to detect a defect in a periodic pattern on the substrate with high accuracy.