Xiaolangdi Reservoir is the largest hydraulic engineering project in the Yellow River Basin, and the annual water-sediment regulation can reduce siltation in the downstream river. However, reservoir storage will change the hydrodynamic conditions of the river. Exploring the differences in the spectral characteristics of dissolved organic matter (DOM) before and after reservoir storage is crucial to reveal the changes in the aquatic ecosystem of the Yellow River Basin. In 2021, water samples were collected before, during, and after the water-sediment regulation period. The characteristics of the change in DOM fluorescence spectral parameters in the reservoir were analyzed. The DOM components and their sources were resolved using three-dimensional fluorescence spectroscopy coupled with the parallel factor method. Combined with the water quality parameters of the water body, the key factors affecting the changes in DOM fluorescence characteristics in the reservoir were elucidated by correlation analysis and redundancy analysis (RDA). Results indicated that (1) the fluorescence index and humification index of DOM did not change remarkably in all periods. The DOM of the reservoir water body was characterized by strong authigenicity and weak humification. During the water-sediment regulation, the authigenic index and freshness index of DOM decreased remarkably, and the newly authigenic DOM produced by microbial activities and other activities decreased in the reservoir area. (2) Four fluorescent components were resolved in the DOM of the Xiaolangdi Reservoir. Differences in the composition of the components were observed in different periods. The C1 and C2 components were humus-like substances, and the C3 and C4 components were protein-like in the non-water-sediment regulation period. On the contrary, the C1, C2, and C3 components were humus-like substances, and the C4 component was a protein-like substance in the water-sediment regulation period. Reservoir DOM before water-sediment regulation was dominated by protein-like substances (52.95%). The exogenous DOM entered the water body because of reservoir drainage and sediment discharge during the regulation period, which led to a remarkable increase in the proportion of humus-like substances (78.53%). The proportion of humus-like substances after water-sediment regulation was 58.80%. (3) Correlation analysis showed that water temperature (WT), flow, and sand content were the main factors influencing the source of DOM in Xiaolangdi Reservoir. During the non-water-sediment regulation period, DOM concentration was mainly affected by the exogenous input of humus-like substances. The degradation of tyrosine components may be related to pH and electrical conductivity(EC). RDA found that pH, WT, and EC were the main environmental factors affecting the differences in DOM fractions in different periods. The results of this study can provide basic data for the study of the fluorescence characteristics of DOM in the Yellow River Basin reservoirs, as well as the scientific basis for further revealing the influence of water body disturbance on the carbon cycle in the Yellow River Basin.