In this paper, the effect of ozone on membrane fouling of membrane bioreactor (MBR) systems was investigated. Sludge specification such as soluble microbial product (SMP), extracellular polymeric substance (EPS), viscosity, particle size distribution (PSD), microscopic observations and sludge volume index (SVI) in presence of ozone were determined. Further, EPS, Fourier-transform infrared (FTIR) analysis, excitation-emission matrix (EEM) fluorescence spectroscopy, and gel permission chromatography (GPC) are considered to determine cake layer characteristics. The results indicated COD removal did not show significant difference with ozonation. Ozone also increases the particle size and decreased protein fraction in EPS and SMP and also resulted in reduction of total EPS and SMP. The obtained results from FTIR spectra show that the relevant group in the cake layer were typically aromatic rings, carbonyl in proteins, carboxyl in humic acids, and N-H stretching in polysaccharides. Further the EEM results showed that the Excitation (Ex) / Emission (EM) = 320/410 peak in the reactor without ozone with intensity of 210 is associated to humic acids, but the peak of humic acids in the reactor with ozone has the intensity of 157 at Ex/Em = 350/450. This indicated the peak shift in the peak. These observations show that the humic acids' structure changed in these two reactors. Also the results show that protein and poly-saccharides on the cake layer reduced in the reactor with ozone but characterization of them did not have any change. Furthermore, the GPC results show that ozone didn't effect on low molecular weight ( < 1 kDa) substances.