Engineered nanomaterials (ENMs) would be settled and accumulated in sediments beneath the surface water. Investigation on the interactions between ENMs and dissolved organic matters (DOM) in sediments could assist researchers in better understanding the environmental behaviors of ENMs. This study adopted heating methods to extract DOM from the sediments in a river of Shanghai, China, and titanium dioxide nanoparticles were used as model ENMs. Three-dimensional fluorescence spectra revealed that humic-like substances possessed a better binding ability with ENMs than protein-like substances and fatty acids. Nano-TiO2 could destroy the α-helix structure of proteins. The rapidly diminishing intensity of α-helix bands suggested that secondary structure of protein was distorted, and the nano-TiO2 binding site was amino residues in main peptide chain. X-ray photoelectron spectra further not only demonstrated that protein-like, humic-like substances and fatty acids could adsorb on the surface of nano-TiO2, but also confirmed that phosphonate could combine with nano-TiO2 via P-Ti covalent bond. This study supported comprehensively scientific data and assisted in understanding the environmental behaviors of ENMs, in order to direct the pollution control of river sediments.