The invention discloses a calibrating method and device applied to different thicknesses of substrates in a laser imaging system. The calibrating device comprises a working platform, a gantry, a calibrating ruler, an oblique cushion block, image collecting equipment CCD and a single-axis displacement platform. A suction disc is placed on the working platform. The calibrating ruler is placed on the oblique cushion block and is provided with equidistant fixed patterns. The oblique cushion block is placed on one side of the suction disc. The gantry is arranged on the working platform. The CCD is slidably connected with the gantry through the single-axis displacement platform. The calibrating device is simple in structure, small in measurement error and strong in practicability; calibration of measurement changes of the CCD can be achieved through simple structures, offset errors, generated in the image collecting process, of the CCD are worked out through measurement results, and the displacement platform of the CCD can be compensated.