An apparatus and method for detecting an alignment mark on a substrate using electron beams. The method include the steps of setting an accelerating voltage of the electron beams in accordance with the layer structure of the substrate, irradiating the substrate with the electron beams having the accelerating voltage set in the setting step, and detecting one of radiation and electrons from the substrate after the irradiating step is performed, and determining the position of the alignment mark based on the detecting operation. The apparatus includes a device for setting such an accelerating voltage, a device for irradiating the substrate with the electron beams, and a detector for detecting one of the radiation and the electrons. In one embodiment, fluorescent X-rays are detected, while in another embodiment secondary electrons or backscattered electrons are detected.