By taking the vegetation map in the 1970s a reference ecosystem and using runoff volume and runoff coefficient as the characteristic indexes of ecosystem runoff regulation function, a runoff regulation function evaluation model based on the SWAT (Soil & Water Assessment Tool) model and a historical reference system was established. Then, the temporal and spatial variation characteristics of runoff regulation function in the Three Headwater Region from 2000 to 2017 were analyzed. Besides, the ratio of the reference runoff coefficient to the actual runoff coefficient was used as the runoff coefficient quality index to describe the deviations of the actual ecosystem from the reference system, for further analysis of the variation characteristics of runoff deviations between the reference ecosystem and the actual ecosystem. Results showed that, between 2000 and 2017, the perennial average runoff and surface runoff were 495.15×10⁸ m³ and 96.64×10⁸ m³, and the perennial average reference runoff and surface runoff were 468.37×10⁸ m³ and 68.60×10⁸ m³. Compared with the reference ecosystem, the runoff storage capacity of ecosystem decreased, resulting in higher runoff and surface runoff. Spatially, the runoff was high in the southeast and low in the northwest, but surface runoff was high in the west and low in the east whether under the reference or actual ecosystems. In terms of the interannual change, we found a visible increase of runoff, especially surface runoff under the actual ecosystem. The difference rate between the actual runoff and the reference runoff ranged from 4% to 9%, and it showed a more pronounced deviation of 22% to 58% of surface runoff. Fro the runoff coefficient quality index score, the average score of runoff coefficient quality index was 98.63, and the average score of surface runoff coefficient quality index was 96.98 in 2000-2017 in the Three Headwater Region. Generally, both scores showed a trend of first decreasing and then increasing, indicating a process of degradation and then recovery during this period, and the change of surface runoff coefficient quality index was more obvious. The scores of runoff coefficient quality index and surface runoff coefficient quality index varied greatly among regions, but the interannual changes were small, with a change of -1.95% to 0.71%, and a change of -0.35% to 1.90%. By analyzing the temporal and spatial variation characteristics of runoff regulation function based on a historical reference system, the comparison of runoff regulation functions in different ecosystems at different times and in different regions was realized, which could provide support for quantifying the process of ecosystem restoration.