This study investigated extracellular polymeric substances (EPSs), including soluble EPS, loosely bound EPS and tightly bound EPS (TB-EPS), in biofilms growing on different carriers from a CANON system to elucidate their different compositions and characteristics. The zeta potentials of all EPS fractions of the two samples were decreased systematically. The soft combination packing (SCP) was more hydrophilic than activated carbon fiber (ACF), and it obtained a significantly higher biomass. However, Raman spectroscopy and Fourier transform infrared spectroscopy revealed the same EPS fraction had similar functional groups between two carriers. Especially for the TB-EPS, total amount and the contents of proteins and polysaccharides in the SCP sample were much higher than those in the ACF sample. Moreover, the TB-EPS fraction of the SCP sample had better bioflocculation activity compared with that of the ACF sample. Therefore the result demonstrated that the characteristics of EPS varied from carrier to carrier.