The adhesion and aggregation properties of microbial cell are closely related to extracellular polymeric substances (EPS). In this work, the composition and physicochemical characteristics of EPS in biofilm and suspended sludge (S-sludge) were determined to evaluate their roles in microbial cell aggregation. Raman spectroscopy and three-dimensional fluorescence spectra have been employed to reveal each EPS fraction in different composition. The flocculating capacity of each EPS fraction in the S-sludge shows extraordinary activity, comparing its counterpart in biofilm. Microbial cell surfaces present high hydrophobicity and increased zeta potentials upon EPS extraction. In addition, the respective contribution of EPS to cell aggregating was elucidated. The contribution of combined SEPS and LB-EPS was 23% for S-sludge sample, whereas that was negligible for biofilm sample. The contribution of LB-EPS and TB-EPS were 16% and 30% for S-sludge sample, and -6% and negligible for biofilm sample, respectively. Therefore, EPS promoted the S-sludge cells to aggregate, while in contrast, they showed a negligible or negative effect on the biofilm cells aggregating.