Whey proteins have important functional properties as it can bind a wide range of bioactive molecules. In this study, the complexation of β-lactoglobulin with retinol at alkaline pH was investigated. The fluorescence spectroscopy was used to determine the temperature dependent behavior of the β-lactoglobulin-retinol acetate (βLG-RET) ensemble. The pattern of protein unfolding in the temperature range of 25–85 °C was followed mainly considering the exposure of tryptophan (Trp) residues. The βLG-RET complex appeared rather stable in the 25–60 °C range, while further increase of the temperature caused partial unfolding. The anisotropy measurements indicated a more flexible conformation at temperature increase up to 80 °C. In addition, the in silico approach was used to complement the experimental results. Important changes in the interaction surface were observed after performing molecular dynamics simulations. The temperature increase caused important rearrangements of the amino acids of the EF loop involved in the interaction with the retinol molecule, which got twisted. These atomic level events induced a significant increase of the affinity between βLG and retinol. This study offers useful information on the potential use of βLG as a carrier for biologically active compounds in order to obtain food products with desired functionalities.