According to one embodiment of a method for measuring a stacking overlay error, the method may use a differential interference contrast microscope system to measure a stacking overlay mark and focus on one overlay mark of a lower layer overlay mark and an upper layer overlay mark when measuring the stacking overlay mark. Then, the method uses an image analysis scheme to obtain an image of the stacking overlay mark from a photo-detector and obtains a first reference position of the lower layer overlay mark in a direction and a second reference position of the upper layer overlay mark in the direction from the image; and computes the stacking overlay error in the direction according to the first and the second reference positions.