The difference calibration and separation patterns are used to calibrate a single-camera with high precision. Several separation calibration patterns with small size are put on the object plane of the camera. Each pattern has some spot array with high precision. Gaussian curved-surface fitting method is used to get the position of spot on the CCD image plane, and the stability (RMS) of measurement result of the spot position is 1 μm (about 0.0053 CCD pixel). The object plane difference coordinates of the reference points on the same calibration pattern are used to calibrate the camera. The mapping relationship between the object plane and the image plane is established with the biquadratic curved-surface fitting method. There is not need for the distance between the patterns. The integer pattern with large dimension is replaced by the several small difference patterns. The difficulty to manufacture the large pattern is avoided. When the error of the relative angle between the patterns is less than 60″. The experiment results show that the calibrated mean value of residual error is 4.8 μm (about 0.028 CCD pixels).