In order to investigate the effect of temperatures and operating modes on extracellular polymeric substances (EPS) contents, three sequencing batch reactors (SBRs) were operated at temperatures of 15, 25, and 35 °C (R15 °C, R25 °C, and R35 °C, respectively), with two SBRs operated under alternating anoxic/oxic conditions (RA/O and RO/A, respectively). Results showed that higher contents of tightly bound EPS (TB-EPS) and total EPS appeared in R15 °C, while loosely bound EPS (LB-EPS) dominated in R35 °C. In all three kinds of EPS (LB-EPS, TB-EPS and total EPS) assessed, protein was the main component in R15 °C and R25 °C, while polysaccharides dominated in R35 °C. Moreover, compared with RO/A, RA/O was favorable for the production of the three kinds of EPS. Furthermore, three kinds of EPS and their components were augmented during the nitrification process, while they declined during the denitrification process under all conditions except for R35 °C.