Bovine β-lactoglobulin (β-LG) present in milks has been found “in vivo” in complexes with lipids such as butyric and oleic acids. To elucidate the still unknown structure–function relationship in this protein, the structural changes of β-lactoglobulin variant A (β-LG A) in the presence of cationic surfactant such as dodecyltrimethyl ammonium bromide (DTAB) have been investigated using various experimental techniques such as UV–vis spectrophotometry, fluorimetry, isothermal titration calorimetry (ITC) and circular dichroism (CD). Subsequently, the retinol binding by β-LG has been investigated in the presence of various amounts of this surfactant as its extrinsic functional binding fluorophore. Comparison of the results allowed to determine the binding of retinol by β-LG in the presence of DTAB. The results of UV–vis and fluorescence studies showed a red shift in wavelength and an increase in absorbance and enhancement in the intensity of the quantum yield of protein during its interaction with DTAB. The results of UV–vis also showed two distinct conformational changes corresponding first to precipitation and second to solubilization of the precipitated β-LG at pH 6.7 and 8.0. The results indicate the cooperative character of binding at pH 2.0. The results of fluorescence studies showed that the binding strength of β-LG/DTAB complex increases with the increase of the pH. CD results showed the shifts in positions of the major minima and change in magnitude of ellipticity and subsequently signified two significant changes in structure of β-LG between 10–30 and 50–100 molar ratio of [DTAB]/[β-LG]. ITC measurements indicated the endothermic nature of β-LG/DTAB interactions at pH 6.7 and the exothermic nature of β-LG/DTAB interactions at pH 8.0. The analysis of the binding data demonstrates the absence of significant changes in retinol-binding properties of β-LG in the presence of various amounts of this surfactant. This implies that surfactant binding does not change the conformation of β-LG in the regions defining retinol-binding site nor interferes with retinol binding by a competition for the same binding site(s)