In our previous study thermodynamic denaturation of bovine β-lactoglobulin variant A (BLG-A), has been investigated in the presence of cetylpyridinium chloride (CPC) as a cationic surfactant. Here, the retinol binding property of BLG was determined at 298 K and pH 8.0 by spectrofluorimeter titration method, in the presence of CPC to elucidate the still unknown structure–function relationship in this protein. Comparison of the results allowed determining the binding of retinol by BLG in the presence of CPC. The two-way chemometrics method was used, to estimate the equilibrium concentration of components by analysis of fluorescence emission spectrum, in order to obtain its equilibrium concentration. The results indicate that the retinol binding properties of BLG do not show significant changes in the presence of this surfactant.