Environmental behavior, bioavailability, and risks posed by Fe3O4, magnetic nanoparticles (Fe3O4 NPs) in surface waters are affected by complex geochemistry, including pH and inorganic and organic matter. This work provides a systematic analysis of adsorption of fulvic acid (FA) on surfaces of Fe3O4 NPs with adsorption kinetics, adsorption thermodynamic, and adsorption isotherm. Adsorption of FA on surfaces of Fe3O4 NPs is consistent with assumptions of Langmuir and Freundlich adsorption isotherm models. The adsorption amount of FA was inversely proportional to solution pH, and the maximum amount is 128.6 mg g−1. Adsorption of FA on surfaces of Fe3O4 NPs is a spontaneous endothermic process. FA plays an important role in aggregation and suspension/sedimentation behavior of Fe3O4 NPs in aquatic environmental. With continuous adsorption of FA, electrostatic repulsion between the particles and the steric hindrance of FA significantly decreased aggregation and increased suspension of Fe3O4 NPs. The results of FTIR and XPS indicated that FA was adsorbed on Fe3O4 NPs mainly through chemical reactions, and carbohydrates particularly play an important role in adsorption.