Changes in phytoplankton community composition can effectively change the composition characteristics of dissolved organic matter (DOM) in water. By studying the spectral characteristics of DOM and the response relationship of phytoplankton community in Xiaolangdi Reservoir of the Yellow River, this study aims to provide a scientific basis for water resources management and water ecological environment protection in the reservoir area. Three-dimensional fluorescence spectroscopy combined with parallel factor analysis was used to analyze the spectral characteristics and sources of DOM. To explore the structural characteristics of phytoplankton community, the diversity index was used to evaluate the water quality of the reservoir area, and the key environmental factors affecting phytoplankton community were explored by redundancy analysis. Correlation analysis was used to explore the relationship between DOM composition and phytoplankton community. The results showed that a total of 110 species of phytoplankton in 6 phyla, 73 genera were identified, and the community structure and relative abundance of phytoplankton were mainly found in the phylum Chrysophyta, Bacillariophyta, and Chlorophyta. Spatially, the relative abundance showed obvious spatial heterogeneity in the community characteristics before the middle reaches>in front of the dam>in the upstream>behind the dam. There was obvious spatial heterogeneity in the community characteristics. The results of diversity index evaluation showed that the water quality in the reservoir area was medium pollution type, and the diversity index in the middle and before the dam was significantly higher than that in the upstream and behind the dam, and the community structure was more complex, indicating that the biological activity of phytoplankton was enhanced after dam construction. The results of redundancy analysis showed that chlorophyll a, total organic carbon, total nitrogen, dissolved oxygen, and pH were the main driving factors affecting phytoplankton community structure in the reservoir area. DOM is mainly composed of humus (C1) and tryptophan (C2) in the ultraviolet region, which are affected by the comprehensive influence of terrestrial input and endogenous contribution, and contribute greatly to the endogenous such as algae and phytoplankton. Correlation analysis showed that phytoplankton community structure was positively correlated with protein-like substances, and dominant species were significantly positively correlated with biological index, indicating that phytoplankton in the reservoir area had an important contribution to DOM.