Purpose: This study describes a two-photon laser fluorescence microscopy technique developed to evaluate the interfacial micromorphology of the hybrid layer in bonded restorations. Materials and Methods: Micropermeability of the hybrid layer was characterized by means of simultaneously contrasting a dye-containing adhesive with a differently colored dye placed into the pulp chamber and allowed to diffuse toward the different-colored hybrid layer. A fluorescent red dye (rhodamine B) was incorporated into a commercial dentin bonding agent. Class I preparations (margins in enamel) were made on extracted human third molars. The teeth were restored using conventional methods: bonding agent, composite, finishing, and polishing. An aqueous solution of a yellow/green dye (fluorescein) was then placed into the pulp chamber for 3 h, allowing time to diffuse toward the different-colored bonded interface. The teeth were then embedded, sectioned, and microscopically analyzed using two-photon laser microscopy at 40X magnification. Results: Subsurface fluorescent imaging using this technique enabled interfacial micromorphology to be characterized at submicrometer resolution and provided high-contrast images. The quality of surrounding structures and potential presence of gaps were also precisely assessed. Conclusion: Two-photon laser microscopy provided high quality, high-resolution images of the bonded interface and surrounding areas, allowing accurate qualitative and quantitative analysis of the structure and integrity of the hybrid layer. Palabras clave: fluorescence microscopy, two-photon laser scanning microscope, fluorescent dyes, resin-based bonded interface