Natural organic matter (NOM) plays an important role in the iodine mobilization in the groundwater system. In this study, the groundwater and sediments from iodine affected aquifers in the Datong Basin were collected to perform chemistry analysis and molecular characteristics of NOM by Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). Total iodine concentrations in groundwater and sediments ranged from 1.97 to 926.1 μg/L and 0.001–2.86 μg/g, respectively. A positive correlation was observed between DOC/NOM and groundwater/sediment iodine. FT-ICR-MS results showed that the DOM in the high-iodine groundwater system is characterized by less aliphatic and more aromatic compounds with higher NOSC, indicating the features of more unsaturated larger molecule structures and more bioavailability. Aromatic compounds could be the main carriers of sediment iodine and were easily absorbed on amorphous iron oxides to form the NOM-Fe-I complex. More aliphatic compounds, especially those containing N/S, experienced a higher degree of biodegradation, which further mediated the reductive dissolution of amorphous iron oxides and the transformation of iodine species, thereby causing the release of iodine into groundwater. The findings of this study provide some new insights into the mechanisms of high-iodine groundwater.