The interaction of the highly cross-reactive anti-PAH monoclonal antibody with four diastereomeric benzo[a]pyrene tetrols (BPTs) is studied by means of fluorescence line-narrowing spectroscopy. It is shown that the interaction of enantiomers of cis-BPT and trans-BPT with the antibody involves different complex geometries. These spatially different ligand-protein interactions alter the relative intensities of the excited-state vibrational frequencies of immunocomplexed molecules allowing for unambiguous spectroscopic resolution of all four enantiomeric isomers. This study represents the first example of a high-resolution, fluorescence-based spectroscopic method capable of enantiospecific differentiation.