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The changes of protein secondary structures in the extracellular polymeric substances (EPS) extracted from activated sludge by four different methods were studied by analyzing the amide I region (1700-1600 cm?1) of the Fourier transform infrared spectra and model protein test. The results showed the molecular weight distribution of organic matter extracted by centrifugation, heating and cation exchange resin (CER) was similar, while the EPS extracted by centrifugation (Control) and CER had similar fluorescent organic matter. The protein secondary structures of extracted EPS by the four methods were different. The similarities of protein secondary structures between the EPS extracted by CER with the Control were the highest among the four extracted EPS. Although the EPS yield extracted by formaldehyde + NaOH method were the highest, its protein secondary structures had the lowest similarity with those extracted by the Control. Additionally, the effects of centrifugation and CER extraction on the secondary structures of bovine serum albumin were also lower than that of other extraction processes. CER enables the second maximum extraction of EPS and maximum retention of the original secondary structure of proteins.