The interactions between chloramphenicol and lysozyme were studied using fluorescence, UV/vis and circular dichroism spectra. The results proved the mechanism of fluorescence quenching of lysozyme by chloramphenicol is due to the formation of lysozyme–chloramphenicol complex. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction, were calculated to be −12.41kJmol −1 and 37.99Jmol −1 K −1 , which indicated that hydrophobic force and hydrogen bond were the dominant intermolecular forces in stabilizing the complex. The distance r=3.99nm between donor and acceptor was obtained according to Förster's theory. In addition, the alterations of lysozyme secondary structure in the presence of chloramphenicol were confirmed by the evidences from circular dichroism, synchronous and three-dimensional fluorescence spectroscopy.