The Tarim River Basin is one of the most ecologically fragile regions around the world in the arid areas of Northwest China. The study of natural vegetation ecological water requirement (EWR) is the basis for the promotion of regional ecological conservation and sustainable development of ecosystems when extreme environmental events occur frequently, which is of great significance for the formulation of scientific and rational ecological conservation strategies. In the study, we improved the vegetation EWR calculation method by introducing a dynamic soil moisture limitation coefficient (KS) and a dynamic vegetation coefficient (KC) that is coupled with a resistance correction factor (Fr) based on the Penman-Monteith method and analyzed its spatio-temporal variation characteristics. Additionally, this study utilized the latitude of ecosystem resilience (LER) to clarify the thresholds for vegetation EWR throughout the growing season in the study area and to analyze the water surplus and deficit (WSD) at different threshold levels. The results of the study show that: (1) Over the past 21 years, the EWR for vegetation has shown a downward trend, with the change in EWR for arbor-shrub forests being more significant than that for grasslands. The average EWR for arbor-shrub forests and grasslands is 36.76 × 108 m3 and 459.59 × 108 m3, respectively. (2) The minimum ecological water requirement (EWRmin) and optimal ecological water requirement (EWRopt) for natural vegetation were 360.45 × 108 m3 and 550.10 × 108 m3, respectively. (3) In EWRmin conditions, the alpine plateau area as a whole showed a water surplus, and the plains area as a whole was in a state of water scarcity, but the precipitation in the study area as a whole could meet the basic survival needs of the vegetation. (4) In EWRopt conditions, the plains and local alpine plateau areas are in a state of water scarcity, the area of water scarcity is gradually increasing, and the regional precipitation is unable to fully realize the objectives of ecological conservation and vegetation restoration.