According to the "Guide for the Demarcation of Ecological Red Line", this paper first demarcated the ecological red line area of the Tarim River basin based on the evaluations of the importance of ecosystem service function and the sensitivity of ecological environment. Then the ecological benefits after implementing the ecological red line policy was evaluated through the FLUS scenario simulation, ecosystem service value (ESV) estimation, landscape pattern index calculation and spatial autocorrelation analysis. The results show that:(1) The total area of the demarcated ecological red line area is 133721 km², accounting for 13.01% of the total watershed area. Among them, the most sensitive areas of land desertification and the most important areas of biodiversity conservation accounts for 60.21% and 30.59%, respectively. The net primary productivity (NPP) of vegetation in the ecological protection red line area accounts for 40.81% of the total in the study area. The average value of NPP within the red line is 4.61 times that outside the line. The demarcation is in line with the principle of "a larger service in a smaller area". (2) The ecological red line area provides 44.55% ESV of the whole basin. The contribution values of the other ecological service functions except hydrological regulation function are all more than 50%. From the perspective of ecosystem service value structure function, regulating and supplying services are the most important functions of the ecosystem; From the perspective of ecological function regionalization, glaciers and lakes contribute 52.28% ESV to the red line area, which plays a vital role in the ecosystem service function in the study area. (3) Through the comparison of ecological landscape pattern scenarios under different development goals, it is found that after the implementation of ecological protection red line policy scenario of returning farmland to forest or grassland and protecting key ecological function areas, the ESV in 2030 will increase by 11.96% over 2015. The area with high ESV will increase by 5.95% and the area with high ESV and low-risk will increase by 6.37%. Compared with 2015, the watershed ecosystem will be more stable in 2030 with a higher capability against risks, and the configuration of ecosystem structure is better. The most important high-ESV-high-risk areas and most of the high-ESV-low-risk areas for the ecosystem function of the Tarim River basin were delimited within the ecological protection red line. Therefore, the ecological red line policy can provide a direction and theoretical support for both the ecosystem stability in the Tarim River basin and the sustainable development of "To gain gold and silver mountains, as well as clear water and green mountains".