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In recent years, the ecological environment along highways in Tibetan Plateau has been severely affected due to the rapid construction of highways. In order to solve the problems of multiple indicators and inconsistent criteria in the ecological impact assessment of highways, and to scientifically screen assessment indicators, the paper proposes a multi-round indicator screening method, which combines literature analysis, expert rating, and statistical analysis. Based on this screening method, normalized difference vegetation index, land surface temperature, elevation, and normalized difference soil index are screened out. Combined with multiple linear regression, an ecological impact assessment model is established and applied to ecological impact assessment of Gonghe-Yushu Expressway. The results show that the expressway construction is the first driving force for the deterioration of the ecological environment along the roadside, and its interference range on the desert grassland ecosystem is greater than that on the agroforestry system. The ecological environment within 150 m on both sides of the expressway should be protected.