Biophysical insight into the binding interaction between the major whey protein, beta-Lactoglobulin (beta LG) and vitamin B12, was studied using different spectroscopic tools such as steady-state & time-resolved fluorescence spectroscopy, Circular Dichroism (CD) and Fluorescence Correlation Spectroscopy (FCS). The intrinsic fluorescence of beta LG was quenched by vitamin B12. From the time-resolved fluorescence experiment, the nature of quenching was found to be static suggesting ground-state complex formation between beta LG and vitamin B12, which was also supported by the excitation spectra. Synchronous fluorescence spectra revealed that the tryptophan residue microenvironment of beta LG was affected by the vitamin B12. The CD spectra suggested that the secondary structure of the beta LG remains unaffected by vitamin B12. From the FCS experiment, the tertiary structure of beta LG was observed to be stable in the presence of vitamin B12 at the single-molecule level. The outcome of this study might have potential applications in the food and pharmaceutical industry.