This paper discusses the effect of dissolved oxygen (DO) on the biofilm structure in membrane bioreactor (MBR) and their consequence on membrane permeability and EPS. Two MBRs under high DO (6.0 mg/L, HDO) and low DO (< 0.1 mg/L, LDO) were operated in parallel under same hydrodynamic conditions. The microbiological aspects in MBR systems were explored through a series of analysis techniques including PCR-DGGE, gel filtration chromatography (GFC), confocal laser scanning microscope (CLSM), and image analysis. The rate of membrane fouling for the LDO MBR was 5 times faster than that for the HDO MBR. The microbial communities between HDO and LDO MBR were quite different, which is likely to be the reason for different structures and permeabilities of the biofilms. The specific biofilm resistance in HDO MBR was lower to that in LDO MBR. This is attributed to relatively lower porosity and higher amount of EPS for the biofilm in LDO MBR. The distributions of cell and EPS were not uniform in the biofilms in both HDO and LDO MBR. The biofilm in LDO MBR contained larger amount of EPS than that in HDO MBR. The ratio of protein to polysaccharide was also higher for biofilm in HDO MBR than in LDO MBR.