Anthropogenic activities and climate change have caused physical and ecological changes in lakes and have aggravated water level fluctuations, which are essential factors to consider for nutrient import, ecological protection, and biodiversity maintenance. Maintaining water levels within a reasonable range is essential for maintaining lake function and ecological health, because ecosystem stability is compromised when water level fluctuations exceed specific thresholds. Thus, the ecological water level (EWL) is an important index for maintaining aquatic habitats and biodiversity. A method for quantifying the EWL of lakes based on hydrological statistical analysis was constructed to bridge the gaps in existing studies, considering both the hydrological alteration and spatio-temporal heterogeneity of water level fluctuations. Taking Poyang Lake as an example, which has recently attracted increasing global attention owing to its water level alterations and subsequent ecological problems, the applicability of the method and rationality of the results were verified. The results indicate that hydrological alteration occurs at the water level of the representative stations, which is jointly affected by climate change and anthropogenic activities in this region. For instance, the construction and operation of the Three Gorges Project affected the water level alteration at Hukou and Xingzi station, and drought further aggravated that of Xingzi station. The calculated EWL of Poyang Lake showed obvious spatiotemporal heterogeneity, which is consistent with the topographic, geographical, and climatic characteristics of the basin. And the rationality of the EWL in this study was verified through literature reviews and satisfiability of characteristic species requirements. The proposed EWL calculation method is simple and feasible with easy data acquisition, strong universality, and broad application prospects, offering a scientific basis and quantitative reference for water resource management and lake ecosystem protection.