The invention provides an alignment device for an inspection substrate, which can pre-align with the large-scale inspection substrate reliably. The device comprises an X-theta shaft workpiece carried stage for supporting the inspection substrate to move in the X-shaft direction and rotate around the theta-shaft direction, a Y-Z shaft contact carrying stage independent of the X-theta shaft workpiece carried stage to be erected along the Y-shaft direction, and a controller for controlling the X-theta shaft workpiece carried stage and the Y-Z shaft contact carrying stage. The controller has such function that, based on the Y-shaft pre-aligning sensor at one side of the Y-Z shaft contact carrying stage and the position information of the inspection substrate detected by at least two X-shaft pre-aligning sensors, the controller controls the theta-shaft rotating mechanism and the X-shaft linear motion mechanism of the X-theta shaft workpiece carried stage to move the workbench in the X-shaft direction and properly rotate the same, therefore the alignment device pre-aligns the inspection substrate.