The conventional methods for calibrating non-uniform of multiple charge coupled deivce (CCD) imaging are difficult and complex because of so many factors leading to imaging non-uniform in a multiple CCD imaging system. Based on analyzing imaging mechanism of multiple CCD, a novel calibration method for multiple CCD imaging using wavelet analysis was proposed. The calibration coefficient was constructed using wavelet transform for calibrating the images captured by different CCD after analyzing the pixel characteristic discrimination of different images. The dynamic calibration efficiency of different wavelets was also discussed in the real-time imaging calibration processing. The result indicated that the correlation value of the images of the same object captured by different CCD was 0.9975 after the calibration. Symlet3 wavelet was more effective than other wavelets both in the two CCD imaging and multiple CCD imaging calibrating. And also it indicated that Symlet serial wavelets were more effective in calibrating the non-uniform of multiple CCD imaging than other types of wavelet. This method provided an effective way for on-line calibrating the multiple CCD imaging system in practical application.