Due to climate change and human activities, the eco-environment quality (EEQ) of eco-fragile regions has undergone massive change, especially in the Tibet Plateau. The Qilian Mountains (QLM) region is an essential ecological function zone in the northeastern Tibet Plateau, which plays a vital role in northwestern China’s eco-environmental balance. However, EEQ changes in the QLM during the 21st century remain poorly understood. In this study, the spatiotemporal variations of the EEQ in the QLM were analyzed from 2000 to 2020 using a remote sensing ecological index (RSEI). The EEQ driving factors are identified by the geographic detector, and the spatial influence of critical factors is represented by a geographically weighted regression model. The results show low EEQ in the QLM. From 2000 to 2020, the EEQ initially slightly improved, then deteriorated, and finally gradually recovered. Spatially, the EEQ shows an increasing trend from northwest to southeast. Moran’s I of EEQ remains at around 0.95, representing high spatial aggregation. “High–High” and “Low–Low” clustering features dominate in the local spatial autocorrelation, indicating the EEQ of the QLM is polarized. Precipitation is the dominant positive factor in the EEQ, with a q statistics exceeding 0.644. Furthermore, the key factors (precipitation, distance to towns, distance to roads) affecting EEQ in different periods vary significantly in space. From results we can draw the conclusion that the natural factors mainly control the spatial patterns of EEQ, while the human factors mainly impact the temporal trend of EEQ, the EEQ in the QLM has been significantly improved since 2015. Our findings can provide theoretical support for future eco-environmental protection and restoration in the QLM.