Fluorescence properties of the macrophytes-derived dissolved organic matter (DOM) and the interaction between Cu-binding and macrophytes-derived DOM under the influence of microbial degradation were studied by microbial degradation experiments and fluorescence titrimetric method. Three-dimensional fluorescence spectra combined with PARAFAC analysis showed that macrophytes-derived DOM contained three fluorescence components, i.e., humus-like components C1, C3, and protein-like components C2, and it was dominated by protein- and humus-like components before and after microbial degradation, respectively. 2D correlation spectra (2D-COS) showed that the protein-like substances were more sensitive and had faster degradation rate in the process of microbial degradation compared with the humus-like materials. Meanwhile, with the increasing concentration of Cu²⁺, the intensity of all fluorescence components was significantly quenched before and after microbial degradation. Compared with the protein-like components, the logKa values of humus-like components of macrophytes-derived DOM before microbial degradation was significantly larger, indicating that humus-like components had a higher metal-binding capacity than protein-like components. However, the logKa values of humus-like components in DOM were smaller than that of protein-like components after microbial degradation. Microbial degradation has a significant impact on the spectral properties and metal-binding capacity of macrophytes-derived DOM, which is meaningful to the bioavailability and ecological risks of heavy metals in lakes.