The eco-environmental impact of highway construction is a long-term and cumulative process. Most of the existing studies pay attention to the environmental impact assessment technology of highway construction period and completion acceptance period, which can not fully explain the eco-environmental impact of highway operation period. At the same time, most of the previous studies have a single index and less consideration of the impact on species. Aiming at the highway located in the forest ecosystem in the middle temperate zone, considering the four aspects of species, land use, landscape pattern and ecological function, this paper establishes the post-evaluation index system of the eco-environmental impact of highway construction projects. This paper puts forward the quantitative assessment technology and spatio-temporal scale identification method of the long-term evolution of the impact of highway construction on the ecological environment, and uses the analytic hierarchy process to construct a comprehensive post-assessment system of ecological environmental impact of highway construction projects. Relying on the Changbai Mountain Ring Road, a 10-year comprehensive ecological impact assessment was carried out. Mammals tend to accumulate within 200m of the road, and highway corridors attract bird activities; the animal mortality coverage rates of amphibians and reptiles are 100%, mammals 66.7%, and birds 50.9%. The utilization rate of bridges and culverts is 88% and 42%, and the total utilization rate is 49.3%. The landscape is changing to the dominant type of coniferous and broad-leaved mixed forest, with lower evenness, less diversity and higher degree of natural connection, but the ability of the ecological environment to resist external changes is reduced. The closer the landscape fragmentation is to the highway, the more serious it is, and the lower the diversity is. The time and space scale of post-assessment of eco-environmental impact of highway construction project is in the range of 4-7 years and 200-300m on the road side, and with the increase of operation time, the change of ecosystem along the line decreases year by year and tends to be stable gradually. The research results can provide a method of reference for the follow-up study of highway ecological environment in mid-temperate forest ecological areas.