Many freshwater lakes are distributed in the middle-lower reaches of the Yangtze River basin; they are of great importance as water supply resources and for aquatic eco-system maintenance. The evaluation of lake health and its variation is useful for environmental management of lake ecosystems. Numerous indices related to physical, chemical, and biological integrity are widely applied to assess the health of lake ecosystems. However, few studies have assessed the ecological integrity of lakes using a combination of physical, chemical, and biological integrity metrics in China, especially at a regional scale. Based on a review of previous research, a lake ecological integrity index (LEII) including physical, chemical, and biological (algae, macroinvertebrates, and fish) integrity metrics was developed to evaluate four large lakes in the middle-lower reaches of the Yangtze Basin. Reference conditions were defined mainly based on historical data collected in the 1950s and 1960s, when the status of lake ecological integrity was acknowledged as "good." The final score for the lake ecosystems integrity index was calculated by combining the scores for physical, chemical, and biological integrity metrics. Additionally, the scores were divided into five categories, i.e., excellent, good, fair, poor, and very poor. The LEIIs of Lake Dongting, Poyang, Chaohu, and Taihu were 66, 71, 57, and 57, respectively. Based on the LEII scores, Lake Dongting and Lake Poyang were rated "good" and Lake Chaohu and Lake Taihu were "fair." However, the individual indices showed different status for the four large lakes. For instance, the physical integrity index was higher than 80 for all lakes except Chaohu Lake (71), suggesting an "excellent" status. The highest score for the chemical integrity index was observed in Lake Poyang (71) and the lowest score in Lake Taihu (56). Three were rated as "good." The biological integrity index ranged from 46 in Lake Taihu to 65 in Lake Poyang, two were rated as "fair." The four large lakes suffered from various degrees of damage in physical, chemical, and biological integrity, and these differences demonstrated the effects of human activity on the ecological integrity of the four large lakes over the past 50 years. Consequently, the LEII can be used as a tool to assess the ecological integrity of the four large lakes along the Yangtze River, and the results provide a scientific basis for lake ecosystem restoration and protection.