The aim of this study was to analyze the distribution of printing ink in the paper more precisely by segmenting the ink layer image from the z direction cross section view of paper. The physical cutting method was used to cut the paper samples and a digital microscope with extra depth of field was used to capture the high resolution views of z-direction cross-section of offset samples printed with cyan ink. After transforming the z-direction cross-section view from RGB color model to HIS color model, the ink layer image was segmented using Euclidean distance as the similarity measurement. By using the HIS image and the threshold of three times of T, the optimum segmentation result of ink layer was got and was valuable for the further quantitative research on the penetration of ink into the paper. When the threshold was set four, two or one time of T, oversegmentation or insufficient segmentation occurred. The penetration depth of ink in the paper can be accurately calculated from the segmentation result of ink layer which can be applied to further researches on the Interaction between ink and paper.