Bombyx mori silk fibroin stained with non-cytotoxic fluorescent materials might be excellent candidates for tissue engineering and regenerative medicine. Here we report the microwave-assisted synthesis of two Schiff bases with fluorescent properties in a short time and good yields. These compounds were analyzed by NMR (1H, 13C-NMR, and 2D-NMR experiments), HRMS (ESI-TOF), UV/Vis, fluorescence spectroscopy, and X-ray diffraction studies. The Schiff base molecular structures showed a non-planar arrangement with the C2 point group. Both compounds showed low cytotoxicity on colon carcinoma cells (Caco2, ATCC-HTB-37) at different concentrations (from 1 to 5 μg/mL). Notably, Schiff bases exhibit good fluorescent staining of bombyx mori silk fibroin by the immersion method and analyzed by confocal microscopy. The fluorescent staining ability of the Schiff bases toward the silk fibroin was explored by molecular docking where both compounds were stabilized by intramolecular H-bondings. From the remarkable findings and to our best knowledge this is the first report of Schiff bases as a fluorescent dye of a biomaterial.