Extracellular polymeric substances (EPS) were extracted from sludge and biofilm via the completely autotrophic nitrogen removal over nitrite (CANON) system. Tightly bound (TB)-EPS were extracted using four physical methods, namely, cationic exchange resin (CER), sonication, heating, and steaming. CER was the most effective and most suitable method for extraction among the four methods. Moreover, the ultraviolet-vis spectra of TB-EPS indicated that few cells were destroyed by the CER method. The major component contents of total EPS, proteins, carbohydrates, humic substances, and DNA in sludge were 60.77, 49.84, 21.63, and 9.01 mg/g volatile suspended solids (VSS) and 90.03, 29.01, 15.96, and 10.04 mg/g VSS in biofilm, respectively. The Fourier transform infrared (FT-IR) spectra results indicated differences in the EPS functional groups between biofilm and sludge. The results of the batch experiments showed that the biofilm activity was significantly higher than that of the sludge in the CANON system. Furthermore, biomass activity was probably influenced by the EPS composition and distribution in the sludge and biofilm.