To scientifically and quantitatively evaluate the impact of Three Gorges Reservoir operations on the runoff and ecological characteristics of Dongting Lake, we analyzed daily flow data from four hydrological stations—Chenglingji, Shiguishan, Nanzui, and Xiaohezui—spanning 1988 to 2020. We employed the IHA-RVA method and Shannon index method to assess flow evolution and its ecological effects during the trial operation phase (2003-2008) and the post-trial operation phase (2009-2020) of the Three Gorges Reservoir. Our findings indicate that: (1) During the operation of the Three Gorges Reservoir, the proportions of water flowing into Dongting Lake from the Xiangshui River, Zishui River, Yuanjiang River, and Lishui River increased, while the proportions from the three outlets of Jingjiang River decreased.(2) In the trial operation phase, the overall changes in flow rate and IHA were greater than those in the post-trial phase, with Chenglingji and Shiguishan exhibiting overall changes of 67.27% and 69.55%, respectively.(3) During the trial operation phase, a reduction in the magnitude of downstream flow increases led to a decrease in both the daily flow rate positive difference (R_rate) and the Shannon index. However, in the post-trial phase, adherence to the “Zhicheng Dispatch” rules, which aimed to ensure small and medium-sized floods downstream and increase dry-season water supply, led to an increase in R_rate and a rebound in the Shannon index. These results provide a scientific basis for ensuring water safety and ecological health in Dongting Lake.