The interactions between bovine serum albumin (BSA) and two substituted hydroxychromone derivatives of coumarin, 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-on (C(3)) and 1,3-dihydroxy-7,8,9,10-tetrahy-dro-6H-benzo[c]chromen-6-on (C(1.3)), were investigated by fluorescence quenching spectra and UV-vis absorption spectra. It was proved that the fluorescence quenching of BSA by C(3) and C(1, 3) was mainly a result of the formation of C(3) and C(1.3)-BSA complexes. The Stern-Volmer quenching constants, binding constants, binding sites and the corresponding thermodynamic parameters Delta H (o), Delta S (o) and Delta G (o) at different temperatures were calculated. The results indicated that van der Waals interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing each complex. The detection limits of C(3) and C(1.3) were 5.08 x 10(-7) and 1.11 x 10(-7) M in the presence of BSA, respectively.