Using molecular docking and multispectral analytical techniques, the present work investigates the binding characteristics between hexanal and β-lactoglobulin at near neutral pH and ambient temperature. Fluorescence quenching demonstrates that binding between β-lactoglobulin and hexanal is of low to medium strength, having a KA ranging from 9.6 × 102 to 4.0 × 104 M−1, with UV–vis and MALDI-TOF analysis suggesting that interactions are non-covalent in nature. Analysis of secondary structure using both FTIR and CD, shows that the complexation of the ligand with the protein induces an increase in β-sheet structure and a corresponding decrease in α-helical components. The method of continuous variation reveals a 1:1.5 (2:3) binding ratio under the present experimental conditions, suggesting that each β-lactoglobulin dimer may bind 3 hexanal molecules. Molecular docking simulations were consistent with these findings, showing three potential binding locations with distinct binding energies for the dimer. One binding site is located within the hydrophobic calyx of each monomer, while the third site is located at the interface between the two monomers. Knowledge achieved presently may advance our understanding of the fate of flavour compounds in dairy based beverage formulations. 3D image of best binding pose between hexanal (in yellow circles) and β-lactoglobulin as a dimer (each monomer denoted as A and B) under ambient conditions at neutral pH, with arrows depicting 2D images of the interactions with amino acids of each protein monomer and interface as well as the binding distances, where green lines represent hydrogen bonds, purple lines represent pi-alkyl bonds, pale purple lines represent pi-alkyl bonds and unbound amino acids interact via non van der Waals interactions. Display omitted •Three binding locations for hexanal on dimeric β-lactoglobulin at neutral pH are proposed.•Binding sites are within the calyx of each monomer and at the interface of the dimer.•Interaction within the calyx possesses the highest binding energy.•Binding between hexanal and β-lactoglobulin is non-covalent in nature.