The present invention provides an aligning apparatus for glass substrate to properly align the glass substrate during transfer along a conveyor. The aligning apparatus includes a plurality of rollers, at least a pair of directional beams, at least a pair of positioning devices, a piston, and a detecting device. The detecting device is used to detect whether there is an offset of the glass substrate during the transportation, and the piston will be triggered once an offset is found and determined. The piston will move the positioning device such that a portion of the positioning device which extends to the roller will push the offset glass substrate so as to bring the glass substrate back to the correct position. The present invention also discloses a method to conduct the alignment of the glass substrate. By the disclosure of the present invention, the glass substrate transferred on the rollers can be automatically adjusted and aligned once an offset is found.