Abstract: In order to find out the optimum dye and staining technique, and to quantify the gross adhesive penetration into wood utilizing fluorescence microscopy, an image analysis system was employed to quantify the effect on the fluorescence interface of sliced yellow poplar plywood of different dyes and staining technologies. The optimal dye and staining technology were confirmed amongst a variety of staining efficiency by controlling the different variables. The results indicated that 05% toluidine blue dying the interphase for 30 min gave the sharpest contrast between a cured adhesive and the wood substrate, and illustrated fluorescence tracking could characterize adhesive penetration effectively. Moreover, the percentage of gross adhesive penetration into wood declines increased as the distance increase from the flake surface. While percentage of total object and total penetration area increased with distance from the bondline surface, more far from bonding interface, more area droplet of adhesive occupied and percentage of penetration area could reach above 90% in this research.