Three-dimensional excitation emission matrix fluorescence spectroscopy (3DEEM)was applied to characterize the loosely bound extracellular polymeric substances (LB-EPS)and tightly bound extracellular polymeric substances (TB-EPS)extracted from aerobic sludge in wastewater treatment. The results showed that three fluorescence peaks were identified in three-dimensional excitation emission matrix fluorescence spectra of the LB-EPS and TB-EPS: peak B (λex/λem=270-280 nm/345-360 nm), peak C (λex/λem=330-340 nm/410-430 nm)and peak D (λex/λem=390 nm/450-470 nm). The peak B was attributed to the protein-like fluorophores, peak C to the visible fulvic-like fluorophore and peak D to the humic-like fluorophore. Results of the peak intensity showed that the major component of the LB-EPS and TB-EPS is protein-like organic matter, then followed by (from high to low): fulvic-like organic matter and humic-like organic matter. The effects of both pH and concentration were significant to the 3DEEMs of LB-EPS and TB-EPS, but the degree is not the same. LB-EPS has the particular chemical structures which are different from the TB-EPS.