The ecological water level is the lowest water level to maintain the integrity and variety of a lake ecosystem. For the protection of the biological habitats and the management of water resources, the study in an ecological water level is meaningful. Based on daily water level data of Lake Gaoyou, northern Jiangsu Province, from 1953 to 2013, the ecological water level data is calculated and treated. The mutation tests are carried using Mann-Kendall method and moving T-test method. The water level regulation in 1953-1992 is analyzed. The monthly progress of the lowest ecological level is also deducted using the annual guaranteed frequency method and dynamic calculation method. The starting time and duration of high/low water levels of Lake Gaoyou are calculated accordingly. The key conclusions are as follows:(1) The mutation point occurred in 1997. (2) The lowest ecological water level of high water level period (from July to October) is 5.8 m, and the time that the water level is higher than 5.9 m needs to be 111 days; the lowest ecological water level of low water level period (from December to next March) is 5.1 m, and the time that the water level is lower than 5.3 m needs to be 96 days; the lowest ecological water level is 5.2 m for the other months. (3) The lowest assuring degree of the ecological water level occurs in July of the year, being 60.83%. The lowest assuring degree in 1993-2013 occurs in 1994, being 49.59%. The days of low water levels are not secured.