An image calibration method of the present invention is configured to calibrate the position of observation area in motion image which includes image frames. The step of the image calibration method includes: determining the observation area and acquires central position of the observation area in first image frame of the motion image; determining first unique area, which complies with gradient characteristic, in the first image frame; acquiring first vector value from the central positions of the observation area to the first unique area in the first image frame; finding second unique area in the second image frame of the motion image according to the gradient characteristic; acquiring second vector value from the central position of the observation area to the central position of the second unique area in the second image frame; and calibrating position of the observation area in a third image frame according to the difference between the first vector and the second vector.