The main stream of the Tarim River in China is typical of ecologically sensitive areas that have been heavily disturbed by human activities; as such, the monitoring of the quality of its eco-environment constitutes an important task for researchers. By using GlobeLand30 data and applying the disturbance degree model and revised ecosystem service value (ESV) model, the study presented in this paper undertook a quantitative estimation of the effects of the disturbance impacts of human activities on the eco-environment of this area in the period of 2000 to 2020. The main conclusions are as follows: (1) disturbance index values, which reflect disturbance to the local ecosystem by human activities, increased over the study period. Further, cultivated land experienced the largest increase, which, in turn, brought about the most significant disturbance to the eco-environment. High disturbance index values presented a patchy distribution in the west of the mainstream of the Tarim River and formed bands and dots in the east; the area of land characterized by high and moderate disturbance index values increased, with growth areas taking on a scattered distribution of patches, bands, and dots without significant spatial continuity. (2) The total ESV increased, indicating the quality of the eco-environment improved. The increase of cultivated land offset the increase in ESV, which counteracted the effects of ecological governance measures. Areas with high ESV values were mainly located in the western and central parts of the study area, while low values were found in the middle east and east. Areas with higher increases in ESV were mainly located in the western and the western part of the middle reaches and took on a zonal distribution, while areas of decrease followed a scattered distribution, presenting as dots or patches. Using the quantitative analysis methods and high-resolution remote sensing data to evaluate the changes in the eco-environment was considered as the innovation of this study, and the findings are useful in exploring the influence of human activities on ecosystems and evaluating the eco-environment in the minor watershed of an arid area. This piece of quantitative research contributes to the task of monitoring eco-environmental changes using remote sensing techniques in ecologically sensitive areas.