Cutting is a relative accurate method in the literatures about ink penetration measurement at present. But cutting may bring some damages to paper, which reduces reliability of measurement result. The objective of this experiment was to investigate the influence of coating thickness on ink penetration and characterize the penetration depth through quantitative analysis by Laser Scanning Confocal Microscope (LSCM). Fluorescent ink was used to observe and characterize ink penetration by LSCM. Three-dimensional images of ink pigment penetration were obtained by reconstructing all XY plane images. The results implied that it was reliable and effective method using LSCM to characterize the ink penetration depth and distribution uniformity. It could be concluded that the print quality could be adjusted and improved by increasing the coating thickness.