Dissolved organic matter (DOM) plays an important role in heavy metal speciation and distribution in the aquatic environment especially for eutrophic lakes which have higher DOM concentration. Taihu Lake is the third largest freshwater and a high eutrophic lake in the downstream of the Yangtze River, China. In the lake, frequent breakout of algae blooms greatly increased the concentration of different organic matters in the lake sediment. In this study, sediment samples were collected from various part of Taihu Lake to explore the spatial difference in the binding potential of DOM with Cu. The titration experiment was adopted to quantitatively characterize the interaction between Cu(II) and DOM extracted from Taihu Lake sediments using ion selective electrode (ISE) and fluorescence quenching technology. The ISE results showed that the exogenous DOM had higher binding ability than endogenous DOM, and DOM derived from aquatic macrophytes had a higher binding ability than that derived from algae. The fluorescence quenching results indicated that humic substances played a key role in the complexation between DOM and Cu(II) in the lake. However, because of the frequent breakout of algae blooms, protein-like matters are also main component like humic matters in Taihu Lake. Therefore, the metals bound by protein-like substances should be caused concern as protein-like substances in DOM were unstable and they will release bound metal when decomposed.