[Objective] To explore the optimal extraction conditions of extracellular polymeric substances (EPS) from activated sludge. [Method] The efficiency of five methods (H2SO4, formaldehyde-NaOH, mixing, heating and NaOH) on the extraction of EPS was investigated comparatively. The optimal extraction conditions of the most suitable method were determined. [Result] NaOH method is most effective in extracting EPS with less DNA contamination and shortened extraction period. The optimal extraction condition was pH of 11, extraction time of 10 min and agitation speed of 80-120 r/min. [Conclusion] The determined optimal extraction condition provided theoretical basis for EPS study.