An off-axis alignment system includes, sequentially along a transmission path of a light beam, an illumination module (10), an interference module (20) and a detection module (30). The interference module (20) includes: a polarization beam splitter (21) having four side faces, the illumination module (10) and the detection modules (30) both located on a first side of the polarization beam splitter (21); a first quarter-wave plate (22) and a first reflector (23), sequentially disposed on a second side opposite to the first side; and a second quarter-wave plate (24) and a cube-corner prism (25), sequentially disposed on a third side of the polarization beam splitter (21); and a third quarter-wave plate (26), a second reflector (27) and a lens (28), sequentially disposed on a fourth side of the polarization beam splitter (21). The second reflector (27) is located on a rear focal plane of the lens (28). A center of a bottom of the cube-corner prism (25) is situated on an optical axis of the lens (28). An off-axis alignment method is also disclosed.