Abstract The conjugation between bovine serum albumin (BSA) and three synthetic food colorants like tartrazine (TTZ), sunset yellow (SY) and erythrosine (ETS) was investigated by fluorescence spectroscopy in tris-HCl buffer solution of pH 7.40. The results indicated that TTZ, SY and ETS could form non-covalent compounds with BSA, which led to the static quenching of endogenous fluorescence of BSA. The binding ability followed the pattern: ETS > TTZ > SY. The values of Hill's coefficients were slightly more than 1 in all systems. Thermodynamic parameters indicated that hydrogen bond and Van der Waals played a major role in the binding of synthetic food colorants to BSA; besides, the competitive experiments suggested that the primary binding site for three synthetic food colorants was located at site I in sub-domain II A of BSA. According to Förster's non-radiative energy transfer theory, the binding distances (r) between synthetic food colorants and BSA were much smaller than 7ߙnm. Meanwhile, synthetic food colorants reduced the binding constants between ciprofloxacin hydrochloride (CPFX) and BSA, leading to the change of medicinal efficacy.