The Chishui River Basin is located in the bordering area of Yunnan, Guizhou and Sichuan provinces, which serves as an important ecological barrier in the upper reaches of the Yangtze River, and plays a leading role in preserving natural environments, protecting water resources, and maintaining soil functions. However, the eco-environmental quality in the basin has encountered serious challenges in recent years, and the conflict between eco-environmental protection and economic development becomes increasingly prominent. Therefore, it is particularly important to quantitatively assess the extent of the eco-environmental changes in this basin. The present study acquired Landsat series remote sensing images based on the Google Earth Engine (GEE) platform, constructed a remote sensing ecological index (RSEI) as the assessment index that reflects the eco-environmental quality using principal component analysis, studied the changing trend in the eco-environmental quality using the Sen–Mann–Kendall trend test, analyzed the spatial clustering distribution patterns of the eco-environmental quality, based on spatial autocorrelation analysis, and applied the geographical detector model to determine the impacts of natural and anthropogenic factors on the eco-environmental quality. We further applied the CA–Markov model to simulate and predict the eco-environmental quality of the basin in 2025. The results showed the following: (1) between 2000 and 2020, the eco-environmental quality of the Chishui River Basin had been greatly improved. The average RSEI value increased from 0.526 in 2000 to 0.668 in 2020, and the percentage of areas belonging to the good or excellent quality category increased from 42.65% to 68.48%. (2) The main drivers of the eco-environmental quality included population density, mean annual temperature, land use type and elevation. The interactive effect between these drivers was significantly higher than that of individual drivers, and thus possessed stronger explanatory power for quality differences. (3) It is predicted that in 2025, the eco-environmental quality of the basin will continue to improve, and the proportion of land areas with good or excellent quality will continuously increase. The present study can provide reference value for local environmental protection and regional planning.