Peri-urban ecosystems are among the most intensive areas in terms of competition between different ecosystem components. Dissolved organic matter (DOM) plays a significant role in aquatic carbon cycling. The chemical composition of DOM and associated potential ecological risks in peri-urban aquatic ecosystems are poorly understood. Herein, we used fluorescence excitation-emission matrix and parallel factor analysis (EEM-PARAFAC) to characterize DOM in a peri-urban critical zone observatory watershed in Eastern China. According to the theory of natural disaster risk formation, we calculated the ecological risk of DOM in the peri-urban watershed. Seasonal variation in DOM concentrations was observed, whereas fluorescent DOM concentrations were site-specific across four sub-watersheds. The analysis of DOM absorption properties revealed the presence of DOM components with high aromatic content and large molecular weight in the watershed. Four fluorescent components (two humic-like and two protein-like substances) were identified using the PARAFAC model. Spatial distribution analysis showed that DOM quality was mainly influenced by human activities, and the proportion of protein-like substance (C3) was strongly correlated with anthropogenic parameters. The distribution of optical indices indicated diverse sources of DOM in the watershed. Ecological risk related to DOM was greater in the dry season than the wet season. There was a slight risk in most areas, with an extreme risk in areas experiencing the most intensive human disturbance, which were also extremely or heavily vulnerable. The results emphasize the strong influence of human disturbance on the ecological risk of DOM in peri-urban aquatic ecosystems. Our study provides useful information for ecological risk assessment of DOM that is difficult to obtain using traditional chemical analysis.