Abstract: The bonding interfaces of carbonized bamboo and bleached bamboo were stained and located by using toluidine blue. Then we optimized the gray value diversity and spectral background noise of interface and conducted the contrast experiments by changing stain concentration and staining time by using image analysis technology. The results showed that toluidine blue staining carbonized bamboo was better than bleached bamboo, and they both got the best parameters at 0.5 % fluorescent dye and 30 min staining time. It was also confirmed from the 3D gray image that there was a great difference of the gray value diversity from three-dimensional view between adhesive and substrate, and it is more suitable to calculate the average penetration depth of the bonding interface by the fluorescent staining.