UV-visible (UV-Vis) absorption spectroscopy, fluorescence spectroscopy (FL), dynamic light scattering (DLS) and isothermal titration calorimetry (ITC) were used to study the interactions between bovine serum albumin (BSA) and the three quaternary ammonium surfactants N-dodecyl-N-(2-hydroxyethyl)-N,Ndimethyl ammonium bromide (DHDAB), N-tetradecyl-N-(2-hydroxyethyl)-N,N-dimethyl ammonium bromide (THDAB) and N-cetyl-N-(2-hydroxyethyl)-N,N-dimethyl ammonium bromide (CHDAB). These surfactants quenched the intrinsic fluorescence of BSA, with longer alkyl chains resulting in more significant quenching. This was attributed to static quenching. Further evidence of static quenching was provided by UV-Vis absorption spectroscopy. The particle size of BSA was found to initially increase and then decrease with increasing surfactant concentration. The concentration of surfactant changed the type of interaction mode. This work revealed the mechanism and binding characteristics between surfactants and protein, and provides the basis