The color difference between original and printed copy is affected mainly by ink trapping and optical properties of overprinted ink layer. Since the general expression itself about ink trapping is affected also by the optical properties, the analysis of color difference using the ink trapping only cannot be certain. This study will show a new approaching method for optical analysis of spectral reflectance and the effect of printing sequence on color difference in multi-color overprints under the condition of excluding completely the ink trapping problems by means of using transparent film as a substrate.