The South to North Water Transfer Project, one of the four largest trans-century projects in China, may produce a series of influence on eco-environment in the middle and lower reaches of the Hanjiang River. So it will be of great significance to carry out an overall evaluation of this project. Based on the principle of building evaluation index system discussed in advance, an overall evaluation index system was built to assess the effects of the south to north water transfer project on eco-environment in the middle and lower reaches of the Hanjiang River Basin. After building an index core quantification model, the score of each index was calculated and the weight of each indicator was decided by the Analytic Hierarchy Process (AHP) method. Then weighted summation of each index was used to make the overall evaluation of the project on eco-environment. The result showed that the construction of the project would produce -37.91% ~ -64.46% of effects on the change of eco-environment over the middle-lower Hanjiang River basin, which should have a strong or significant negative impact. And among the six eco-environment variables, the order from strong to weak in the extent was water equality, soil-geological variables, society production, aquicolous biology, water resource and climate respectively. In these variables, the upper effect magnitude of the project on water equality, social production, soil-geological and aquicolous biology variables exceed -40% and on water resource variable also exceed -20%. In terms of the five levels of eco-environmental impacts, including no, minor, significant, strong and extreme impacts, water equality was in the state of extreme or strong negative impact, soil-geological variable belonged to a strong, significant or extreme negative impact and social production belonged to a strong or significant negative impact state, aquicolous biology variables received a strong negative impact, water resource was in the state of significant negative impact, but climate in the minor negative impact state and the magnitude was close to zero.