The source and composition of dissolved organic matter (DOM) are important drivers of its biogeochemical role in aquatic environments. Different land use types may alter DOM amount and composition in freshwaters. Here, water samples were collected from the outlets of 16 subcatchments within mixed land use patterns in the South Tiaoxi River in Eastern China. Dissolved organic carbon (DOC), DOM absorption coefficient (α₃₅₀), and fluorescence spectrum were measured. These 16 subcatchments were grouped into four clusters with different land use features: natural forest land, planted forest land (Phyllostachys praecox, a bamboo species), cropland, and residential land. Two humic-like and two protein-like fluorescent components were identified using parallel factor analysis (PARAFAC). DOC concentrations were the lowest in natural forest land dominated subcatchments as compared to other land uses with intensive anthropogenic activities, so did α₃₅₀ and fluorescent intensities of different components. Protein-like fluorescence was more pronounced for the subcatchments dominated by residential land, while terrestrial humic-like fluorescence was the most abundant component for the subcatchments dominated by the other three land use types. The relationships between the percentage of cropland and that of fluorescence fraction appear to be a threshold response, indicating that effects on DOM composition varied with agricultural activity patterns. The reservoir would have a positive effect on DOM amount and the percentage of protein-like fluorescence associated with autochthonous activities. This study in general shows the impact of anthropogenic land use patterns on the amount and composition of DOM in headwater streams, which may affect ecosystem function and health of aquatic environments.