Abstract: In order to explore the optimal operating conditions and performance of anaerobic biofilters (AF) for treating polluted urban rivers (Xin River), the transformation characteristics of dissolved organic matter (DOM) in a pilot-scale AF at different hydraulic retention time (HRT) and temperatures were studied by using three-dimensional fluorescence spectroscopy and parallel factor analysis (EEM-PARAFAC). The results showed that: (1) DOM in the Xin River mainly contained three fluorescent components (C1, C2, C3), corresponding to humus-like, protein-like and humus-like substance, respectively. Protein-like and humus-like substances accounted for 53.45% and 46.55%, respectively. (2) When the AF was stable, 30.75% COD and 19.80% UV₂₅₄ were removed. Also, the analysis of fluorescent components indicated that the effective removal of C2 and C3 contributed to the transformation of DOM, and the increase of temperature and HRT increased the removal of DOM. (3) UV₂₅₄ and DOM with molecular weight <10 kDa gradually decreased along the filter. The removal rates of DOM with molecular weights 3-10 kDa and <3 kDa were 64.29% and 22.81%, respectively. Moreover, three-dimensional fluorescence spectrum of the effluent along the filter indicated that the microbial activities in the front of AF was higher. C1 and C3 changed slightly, C2 increased at first and then decreased gradually, the fluorescence intensity of DOM in the final effluent decreased significantly. It further demonstrated that DOM removal occurred mainly in the first filter layer of AF. In summary, AF could convert refractory organic matter into easily degradable organic matter and effectively remove DOM in polluted urban rivers under normal temperature and HRT=24 h, and could be used as an effective potential pretreatment to treat polluted urban river.