This paper demonstrated a general algorithm to carry out precision alignment with UVW or XXY and XYθ stages using two cameras without rotation center information. Image processing with template match was implemented to search for fiducial mark under the field of view of a camera as the positioning tool. We established the relationship between cameras and motion stage by calibration procedure with inverse regression method. We finished the substrate’s alignment parallel to the mechanical axis in 2–4 times iterations and applied this algorithm in sixth-generation LCD prober system successfully.