In a method of detecting a defect on an object, a preliminary reference image can be obtained from a plurality of comparison regions defined on the object. The preliminary reference image is divided into reference zones by a similar brightness. Each of the reference zones is provided with substantially the same gray level, respectively, to obtain a reference image. Whether a defect exists in an inspection region in the comparison regions is determined using the reference image. Thus, defects in the inspection regions having different brightnesses can be detected using the properly obtained reference image.