In this paper, we employ atomic force microscopy (AFM), fluorescence recovery after photobleaching (FRAP) technique, phospholipid fatty acids (PLFA) and MiSeq analysis to study the effects of traditional dissolved oxygen (DO) levels (0.71-1.32 mg/L, 2.13-3.02 mg/L and 4.31-5.16 mg/L) on surface force, cell membrane properties and microbial community dynamics of activated sludge. Results showed that low DO level enhanced the surface force and roughness of activated sludge; the medium DO level decreased cell membrane fluidity by reducing the synthesis of branched fatty acids in the cell membrane; high DO level resulted in the highest protein content in the effluent by EEM scanning. Abundance of Micropruina, Zoogloea and Nakamurella increased and Paracoccus and Rudaea decreased with the increase of DO levels. RDA analysis suggested that saturated fatty acids (SFA), anteiso-fatty acids (AFA) and iso-fatty acids (IFA) were closely related to effluent quality as well as some genera. (C) 2016 Published by Elsevier Ltd.