Quantitative assessment of the climate change and human activity to the run-off alteration is significant for the sustainable management of regional water resources. The time series of run-off from 1960 to 2015 are characterized by three periods: the baseline period(1960-1971), the changing periods Ⅰ(1972-1985)and the changing period Ⅱ(1986-2015)according to the trend analysis. The sensitivity and its spatial distribution of run-off alteration on climate changes and human activities are analyzed based on the water balance and the Budyko hypothesis. The results show that the streamflow alteration is most sensitive to human activities in the Tarim River Basin during the period of 1986-2015. An obvious increase of run-off sensitivity occurred on the precipitation, potential evapotranspiration and catchment parameter from the baseline period to the impact period Ⅱ, and sensitivity indexes are in 1. 26, -0. 26 and -1. 88 in changing period Ⅱ, respectively. Moreover, a regional difference has been observed in the elasticity coefficient and the downstream is higher than the upstream of Tarim River. In the view of the deepening and aggravating loss of run-off due to the climatic change and anthropogenic factors, it is significant for the development and management of regional water resources by taking timely measures according to the sensitivity analysis.