An overlay measuring method includes irradiating an electron beam onto a sample, including a multi-layered structure of overlapped upper and lower patterns formed thereon, to obtain an actual image of the upper and lower patterns. A first image representing the upper pattern and a second image representing the lower pattern are obtained from the actual image. A reference position for the upper and lower patterns is determined from a design image of the upper and lower patterns. A position deviation of the upper pattern with respect to the reference position in the first image and a position deviation of the lower pattern with respect to the reference position in the second image are calculated to determine an overlay between the upper pattern and the lower pattern.