Provided are a substrate separation apparatus and method. The substrate separation apparatus includes: an upper transfer part which fixes the upper substrate and transfers the upper substrate in a positive X-axis direction in an XY coordinate system composed of an X axis and a Y axis intersecting each other; and a lower transfer part which is installed under the upper transfer part with a gap therebetween and fixes and transfers the lower substrate, wherein the lower transfer part includes: a lower holding unit on which the lower substrate is placed; and a lower guide unit which includes path guides for guiding movement of the lower holding unit, wherein the path guides include a first path guide which is parallel to the X axis and a second path guide which is continuous with the first path guide and contacts a straight line having a negative slope in the XY coordinate system.