Eysenhardtia polystachya (EP) is an endemic Mexican plant that has been widely studied for its antidiabetic, antibacterial, and antioxidant properties. Several studies had reported the main components of EP, but their fluorescence properties had not been broadly studied. In a previous study we obtained extracts with different composition from this plant and they presented florescence. In this work we study fluorescent compounds from EP and evaluate their fluorescence properties. EP extracts were obtained by Soxhlet extraction with ethanol, samples were dried, and compounds were separated by column chromatography. Fluorescent fractions were classified apart from other fractions and characterized by Scanning electron microscopy (SEM), UV-Vis, Raman, FTIR and H NMR spectra. Additionally, we obtained functional nanomaterials (using silica nanoparticles). TD-DFT molecular calculations of the fluorescent components were carried out to compare their theoretical UV-Vis spectra to experimental results. Nine fractions were obtained by chromatography and five of them showed fluorescence. Fluorescence of extracts from Eysenhardtia polystachya is due to more than one component and we suggest that could be other hydrochalcones for which we present possible structures. This finding would help to dissipate questions about which component is responsible for fluorescence in extracts from the plant and in this way determinate the appropriate use for these fluorophores. Finally, the application and viability as a biosensor using pulmonary epithelium fibroblast cell culture IMR-90 was proved, and in the concentration used are non-toxic materials.