Cross flow ultrafiltration systems and three dimensional fluorescence spectrum were used to investigate the fluorescence characterization of different molecular weight dissolved organic matter in water of wet season and dry season from Yangtze River estuary, in order to find out the sources and influencing factors. The results showed that four types of dissolved organic carbon (DOM) fluorescence peaks were observed in the water: humic-like fluorescence peak A and C, and protein-like fluorescence peak B and D. Fluorescent substances mainly existed in the truly dissolved organic matter (UOM, <1 kDa), secondly in low and medium molecular weight of colloidal organic matter (1~500 kDa). Protein of DOM in water mainly originated from anthropogenic sources, then from autochthonous sources. Compared to protein, humic acid of DOM in wet season was both terrigenous and autochthonous sources, while in dry season mainly from terrestrial organic matter. In addition, humic acid in UOM was mainly derived from biological decomposition, however, in colloidal organic matter (COM) came from terrestrial organic matter and was affected by resuspension effects. There was linear correlation between fluorescence intensity and environmental parameters. It was revealed that the complex environmental conditions would influence the fluorescent substance of DOM in water from Yangtze River estuary.