The factors which lead to non-uniformity of images are very complex. The conventional methods for calibrating non-uniformity of multiple Charge Coupled Device (CCD) imaging can not consider all of these factors. According to the characters of non-uniform images of multi-CCD, a calibrating method based on the scene was proposed by using the theory of two-point calibration and finding out the linearity of the images. Compared with the conventional two-point calibration and multi-point calibration methods, contrastive calibrating results were obtained. This method can calibrate the scene images without standard illumination images, which provides an effective way for online calibrating the multiple CCD imaging system in practical application.