The aggregation process of engineered nanoparticles (ENPs) is important in assessing their fate and transport in the environment. Here, we present the application of isothermal titration calorimetry (ITC) in studying the thermodynamics of ENPs' aggregation in aqueous solutions containing monovalent (NaCl) and divalent (CaCl2) electrolytes, natural organic matter, and hematite natural NPs, which enables us to elucidate their interaction mechanism. The free energies for the aggregation of TiO2 at different solution conditions were dominated by large favorable entropy, presumably because of the expulsion of bound water molecules to the solution upon complexation. The copresence of humic acid and Ca2+ facilitated aggregation for both homo- and heterosystems through intra- or intermolecular bridging, leading to the formation of more compact aggregates. We believe that this ITC strategy can be successfully used to characterize the interaction details between ENPs and various environmental components in ambient water systems.