Three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy was used to investigate the characteristics of dissolved organic matter (DOM) in five typical eutrophic agricultural reservoirs. Based on catchment ecosystem, the five reservoirs were divided into three pollution sources of livestock, living, and farmland sources. The quantities and qualities of DOM in the reservoirs were analyzed. Our results showed that DOM characteristics were different in eutrophic reservoirs based on source. More protein-like components were observed in the reservoirs with the living sources, while more humic-like components were seen in the reservoir with farmland sources. Additionally, correlation analysis showed different sources for protein-like and humic-like components. Protein-like components originated mainly from phytoplankton (endogenous sources), and humic-like components were from terrestrial sources. Furthermore, the high values of specific fluorescence parameters were consistent with a dominant role of endogenous DOM in eutrophic water bodies, with FI values (fluorescence index) of approximately 1.9, and β:α values (freshness index) greater than 0.7. This result indicated that mixed features dominated endogenous sources in the reservoirs, regardless of terrestrial pollution sources. By comparing our fluorescence characteristics and historical references, we confirmed that catchment ecosystems related to human activities are important factors in determination of the characteristics of DOM in aquatic environments. However, complex and extensive eutrophication requires endogenous control of water bodies, which will play a central role in improving water environments and sustainable use of reservoirs. Therefore, this study provides an effective basis for water quality assessment of eutrophic agricultural reservoirs.