The removal of dissolved organic matter (DOM) during municipal solid waste leachate treatment in a membrane bioreactor (MBR) was investigated. The MBR was operated with fouled and cleaned membranes, at a time. DOM, mixed liquor, and effluent were characterized by fractionation and fluorescence excitation–emission matrix (EEM) spectroscopy. Results showed DOM removals of 82% during the MBR operation with the fouled membrane. The DOM characterization indicated that most of the hydrophilic compounds and protein-like substances could be significantly removed by microbial activities in the treatment processes. Furthermore partial removal or transformation of organic substances was observed during membrane filtration. The fouled membrane improved DOM rejection by only about 2% when compared to the cleaned membrane, which demonstrated the significance of irreversible foulants on DOM rejection. There were insignificant differences in DOM fractionation and EEM fluorescence spectra between water samples filtered through the fouled and cleaned membranes.