Anaerobic-anoxic-oxic process with tertiary filtration (A2OWTF) and membrane bioreactor (MBR) was compared from the perspective of dissolved organic matter (DOM) variation in this study. A2OWTF and MBR wastewater treatment plants (WWTPs) samples were compared via conventional wastewater quality indexes, excitation-emission matrix (EEM) spectra, UV–vis spectra and high-performance size exclusion chromatography (HPSEC), which gradually revealed differences between the two processes from phenomena to mechanism. Lower removal of dissolved organic carbon (DOC) by MBR phenomenally revealed its DOM removal deficiency. EEM fluorescence regional integration (FRI), EEM parallel factor analysis (PARAFAC) and UV–vis Gaussian deconvolution qualitatively revealed more abundant humic-like substances (HS) and more complex effluent DOM composition for MBR. HPSEC provided more quantitative characterization. As for effluent after biotreatment, MBR exhibited lower removal rates of HPSEC fractions I (mainly containing protein and polysaccharides), IV (mainly containing low molecular weight organic acids) and fractions II, III (mainly containing HS) than A2OWTF (e.g., removal rates of MBR vs. A2OWTF calculated with UV absorbance at 254 nm (UVA254): 43.6% vs. 66.2%; 5.1% vs. 67.0%; −14.4% vs. 30.8%; −5.7% vs. 36.8% respectively for fractions I, IV, II, III). The massive HS generation in MBR also led to its higher trihalomethane formation potential (THMFP) for final effluent than A2OWTF. In conclusion, MBR should be prudently considered in terms of DOM removal for municipal WWTP upgrading.