A camera calibration method for micro-image measuring system is presented. Firstly the micro image of known size microscopic scale is obtained. Followed by pre-processing and edge detection, ruler lines can be cut out from the image. Then based on Hough lines, the longer ruler lines can be identified. So the calibration factor can be calculated by the physical distance and the corresponding pixel distance of the lines. The calibration objects are existing microscopic scales, so this method is easily applied. The experimental results show that calibration precision is high.