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Abstract: The interactions of BSA with a series of cationic imidazolium gemini surfactants ([C n -s-C n im]Br2, n =10, 12, 14, s =2, 4, 6), quaternary ammonium surfactants (C12C2C12), and their corresponding monomers ([C12mim]Br and DTAB) are investigated by fluorescence using pyrene as a molecular probe, synchronous fluorescence, circular dichroism (CD), and UV–visible absorption spectra. These surfactants are used to elucidate the effects of surfactant hydrophilic head group, spacer length, and hydrophobic chain length on the conformation of BSA. The results of fluorescence spectra and CD show that the imidazolium gemini surfactants with shorter spacers or with longer hydrophobic chains have a larger effect on BSA unfolding, and the imidazolium gemini surfactant interacts with BSA more strongly than its corresponding monomer and the quaternary ammonium gemini surfactant. These conclusions have been confirmed by the binding constants (Ka ) and binding sites (n) for the BSA/surfactant system. Stern–Volmer quenching constants KSV of cationic surfactants binding to BSA are obtained, indicating that the probable quenching mechanism is initiated by ground-state complex formation rather than by dynamic collision. Moreover, the synchronous fluorescence spectra show that the surfactants mainly interact with tryptophan residues of BSA. [Copyright &y& Elsevier]