Abstract Adsorption of Enrofloxacin (ENR) on minerals dominates the fate and transport of ENR in the environment. In this study, the sorption process of ENR on montmorillonite and the impact of dissolved organic matters (DOMs) on ENR-montmorillonite interactions were investigated using in situ ATR−FTIR spectroscopy and two-dimensional correlation analysis (2D-COS). Negative peaks were observed in the 3400–2900cm−1 region due to the loss of hydrated protons at montmorillonite surfaces. The primary characteristic peaks of adsorbed ENR molecules were resolved in the 1800–1100cm−1 range. The results of 2D-COS suggested the sorption process was initiated by the interaction of hydrated protons on montmorillonite surfaces with diverse moieties of ENR molecules depending on pH. The sorption mechanism of ENR was mainly cation exchange at acidic condition, charge neutralization at neutral condition, and proton transfer at alkaline condition. DOM could interact with piperazinyl amine groups of dissolved ENR, which changed the interaction sequence of ENR molecule with montmorillonite surfaces. Electrostatic interaction was the predominant driving force for the interaction between DOM and dissolved ENR. H-donor–acceptor interaction and π−π interaction may also be responsible to this interaction. Insights gained from this study improve our understandings on sorption mechanism of ENR and similar ionic organic pollutants in soil systems.