Two membrane bioreactors (MBRs) with different operation conditions were employed to investigate the role of dissolved organic matter (DOM) in membrane fouling. DOM characteristics and their correlations with membrane fouling in the MBR systems were studied by using three-dimensional excitation-emission matrix (EEM) fluorescence technology, gel filtration chromatography (GFC) analysis, and column chromatographic method for DOM fractionation, etc. The three-dimensional EEM fluorescence spectroscopy analysis indicated that the fluorescence intensity of protein-like peaks in DOM samples collected from the MBR zones showed positive correlations with membrane fouling. The fluorescence spectra of membrane foulants also exhibited two protein-like peaks, confirming that proteins played an important role in membrane fouling. The DOM samples collected from MBR zones were fractionated into four components, i.e., hydrophobic (HPO), transphilic (TPI), charged hydrophilic (HPI-C) and neutral hydrophilic fractions (HPI-N). It was found that HPI-N was the most abundant fraction in all the samples, accounting for 42.0–48.9% of the total DOM. Test results also showed that HPI-N had the highest fouling potential, which could be attributed to the high molecular weight (MW) distribution and the high membrane rejection rate of macromolecules.