Water use efficiency (WUE) is the basis of carbon and water cycle in ecosystem and an important ecological indicator for evaluating vegetation growth conditions. In order to reveal the spatiotemporal changes of WUE of different vegetation types in Manas River Basin, analyze the differences of WUE of different vegetation types, and discuss the driving factors affecting WUE, the WUE of Manas River Basin from 2001 to 2019 was quantitatively estimated based on MODIS data. Moreover, the coefficient of variation, Theil-Sen median trend analysis, M-K significance test and Hurst index were adopted to analyze the current situation and future trend of spatiotemporal changes of vegetation WUE, and geographical detectors were applied to quantitatively analyze the influencing factors of WUE in the study area. The research results show that the WUE in the Manas River Basin fluctuated between 0.74 and 1.08 g/(mm·m2) from 2001 to 2019, and the multi-year average of WUE was 0.88 g/(mm·m2), which showed an overall downward trend, with a decrease of 6.82%. The spatial difference of WUE was significant, showing a distribution pattern of high in the central and northern parts but low in the southern part; high in the middle reaches of the basin but low in the upper and lower reaches. The average WUE of forest land was relatively high among the vegetation types, and the average WUE of coniferous forest was the highest, which was 1.52 g/(mm·m2); followed by broad-leaved forest, which was 1.29 g/(mm·m2). The main driving factors of WUE in the study area were CO2, soil moisture, temperature vegetation drought index and vapor pressure deficit. Compared with single factor, the interaction of multiple factors had a greater impact on WUE in the study area, all the two-factor interaction detection showed a significant enhancement relationship, and each explanatory power was more than 60%, among which the explanatory power of the interaction between CO2 and soil moisture was the highest (90.3%). The research results can provide a technical support for efficient agricultural production, optimal management of water resources and ecological restoration in the study area.