The water-rock interaction has a significant effect on the binding characteristics of dissolved organic matter and heavy metals when mine water flows in goaf. This study used fluorescence excitation-emission matrix (EEM) quenching combined with parallel factor (PARAFAC) analysis to characterize the binding properties of DOM with Fe (Ⅲ), Fe (Ⅱ+Ⅲ) and Mn (Ⅱ) in mine water under rock-influenced conditions. Two protein-like components and two humic-like components were identified by PARAFAC, in which protein-like components dominated (75.9%). The fluorescence intensity of each component can all be weakened, especially the stability constant (logKM) value of Fe (Ⅱ+Ⅲ) with fulvic-like acid and humic-like acid was the largest and the binding was more stable. Clay minerals and iron-bearing minerals in rocks had significant effects on the binding characteristics of DOM and metal ions under water-rock interaction. Iron ions released by the oxidation of pyrite and siderite in sandstone can reduce the fluorescence intensity of the derived components. The competitive adsorption effect of clay minerals on metal ions made the fluorescence intensity of the derived components under the action of sandstone containing less clay minerals (19.5%) be lower than that of mudstone (31.3%). Meanwhile, the process of water-rock interaction was accompanied by microbial activities to convert protein-like components into fulvic-like and humic-like components, or higher levels of stable substances. This study shows that when assessing the potential ability of mine water DOM and metal ions binding and migration during the flow of water in goaf it is crucial to take into account the presence of water-rock interaction. Display omitted •EEM-PARAFAC analysis identified 2 protein-like and 2 humic-like components.•Iron ions from iron mine oxidation reduced the fluorescence intensity of DOM.•Competitive adsorption of clay minerals affected the binding of DOM and metal ions.•The process of water-rock interaction was accompanied with microbial activity.•Water-rock interaction manipulated DOM and heavy metal binding properties.