The interaction characteristics of 2-chlorophenol (2-CP) with catalase (CAT) were investigated by employing fluorescence spectroscopy and UV-Vis absorption spectroscopy. The intrinsic fluorescence of CAT was quenched distinctly by 2-CP. The quenching mechanism of fluorescence of CAT by 2-CP was observed to be a static quenching procedure. The thermodynamic parameters indicated that the binding reaction was spontaneous and the hydrophobic force played the major role in stabilizing the 2-CP-CAT complex. The binding constant was 1.18×10-4 L/mol. The binding distance r and critical distance R0 was 1.90 nm and 1.64 nm, respectively.