A detailed investigation was conducted on the reaction of a 1,1′-bi-2-naphthol–coumarin-based fluorescent probe with amino acids. On the basis of the studies, including fluorescence spectroscopy, ¹H NMR, UV–vis, mass spectroscopy, single-crystal X-ray analysis, and molecular modeling, it was found that the distinctively different fluorescent responses of the probe toward the amino acid at the two excitation wavelengths are due to two different reaction pathways that generate different intermediates and products.