Nanofiltration membrane bioreactor (NF-MBR) was proposed to provide quality effluent free of virus and most large organic substances. The dissolved organic matter (DOM) in soluble microbial products (SMP), loosely bound extracellular polymeric substances (LBEPS) and tightly bound extracellular polymeric substances (TBEPS) of wastewater sludge before and filtered through nanofiltration membrane were fractionated using size exclusion chromatography, characterized with ultraviolet absorbance at 230, 254 and 280 rim, dissolved organic carbon (DOC) content, and excitation-emission-matrix (EEM) spectra. Except for long-chain organic acids, the membrane rejected most organic matters in SMP and LBEPS fractions. However, the membrane failed to reject fluorescent DOM from TBEPS at molecular weight of 3000-7000 Da. The content of TBEPS in the mixed liquor affected significantly the effluent quality in NF-MBR application.