The interaction between umbelliferone (UMB) with tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) was studied by using fluorescence (FS) and ultraviolet (UV) spectroscopy. The results show that UMB can strongly quench the fluorescence of the three aromatic amino acids with the maximum quenching wavelengths at 347, 303 and 282 nm, respectively. Data analyses based on the Stern-Volmer curve and the UV spectroscopy show that static quenching occurred through the formation of the complexes of UMB with each aromatic amino acid in a molar ratio of 1∶1. The binding constant Kc of UMB with Trp, Tyr and Phe is 2.993×106, 7.858×104 and 1.186×103 L·mol-1(298.15 K) and 2.702×104, 1.063×105 and 8.352×103 L·mol-1(310.15 K), respectively. The thermodynamic parameters indicate that UMB has a strong interaction with the three aromatic amino acids. Hydrogen bond and Van der Waals force may play a major role in the reaction of UMB with Trp, whereas hydrophobic interaction should be responsible for the binding of UMB with Tyr and Phe. In addition, the dipole-dipole interaction may be another factor in the reactions between UMB and the three aromatic amino acids.