Steady state fluorescence measurements have been used for the investigation of interaction between the bovine serum albumin (BSA) and fluorescence probes: 3-hydroxy-2,4- bis[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ6), 3-hydroxy- 2,4-bis[(3-methyl-1,3-benzothiazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ7) and 3-hydroxy-2,4-bis[(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)methyl]cyclobut-2-en-1-one (SQ8). The binding constant between bovine serum albumin and squarine dyes has been determined by using both the Benesi-Hildebrand and Stern-Volmer equations. The negative value of free energy change indicates the existence of a spontaneous complexation process of BSA with squarine dyes.