Extracellular polymeric substances (EPS) are the essential components of activated sludge for removal of pollutants from wastewater. Limited information is available on the binding constants and binding mode of organic pollutants to EPS. In the present study, binding of phenanthrene (PHE) to extracellular polymeric substances (EPS) from aerobic activated sludge was investigated using fluorescence spectroscopy. Two protein-like fluorescence peaks (Ex/Em=225nm/340nm for peak A; Ex/Em=275-280nm/338nm for peak B) were identified in the three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy of the EPS. The fluorophores in EPS were clearly quenched by PHE and the quenching processes were static. The quenching constants (lnK(a)) were in the range of 11.27-13.82M(-1) and the binding constants (logK(b)) in the range of 6.11-8.98M(-1). The binding site number increased with increasing temperature. The corresponding thermodynamic parameters DeltaG, DeltaH and DeltaS were calculated. The interaction of the fluorophores in EPS and PHE is spontaneous and exothermic. The binding of EPS with PHE is dominated by the hydrophobic interactions. Fluorophore A has stronger hydrophobic interaction with PHE than fluorophore B.