In this study, we have characterized three fluorescent components of dissolved organic matter (DOM) in the surface and underground water of one rare earth element ore district by excitation–emission matrix (EEM) fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC). Two protein-like components (C1, tyrosine and C2, tryptophan) and one humic-like component (C3) were identified by the DOM Fluor-PARAFAC model, with C3 constituting more than 95% of the total DOM, while C1 and C2 occupying a tiny fraction of DOM. The distribution of three PARAFAC-identified components was strongly influenced by the river direction, terrain and location of various water samples. The results suggested that DOM of samples collected from downstream or a central region had higher fluorescence intensity than those of upstream or surrounding the center. In addition, a negative linear correlation (R² = 0.8465) between pH (5.7–9.2) and fluorescence intensity of C3 was observed, indicating that the increase of pH might enhance the intensity of fluorescent humic-like substances. Although the fluorescence intensity of C1 and C2 was independent of pH changes, strong quenching effects of different heavy metals were presented for C1, and evident positive correlations between C2 and concentrations of rare earth metals (La, Ce, Tb, Dy, Tm, etc.) were observed, which showed that tyrosine-like (C1) and tryptophan-like (C2) substances were assumingly responsible for metal binding and adsorption in water, respectively. Based on EEM-PARAFAC modeling, all the fluorescence EEMs of samples could be decomposed into a three-component model, and their potential applications in water quality monitoring and metal-binding indicator were likely to be developed in the fluorescence analysis of natural water.