It is crucial to improve water use efficiency (WUE) and the transpiration fraction of evapotranspiration (T/ET) for water conservation in arid regions. As a link between carbon and water cycling, WUE is defined as the ratio of gross primary productivity (GPP) and ET at the ecosystem scale. By incorporating the effect of vapor pressure deficit (VPD) on WUE, two underlying WUE (uWUE) formulations, i.e. a potential uWUE (uWUEp = GPP·VPD0.5/T) and an apparent uWUE (uWUEa = GPP·VPD0.5/ET), were proposed recently. Both uWUEp and uWUEa can be estimated from eddy covariance measurements, and the ratio of uWUEa and uWUEp was then used to estimate T/ET. This new method for ET partitioning was applied to three typical ecosystems in the Heihe River Basin. Growing season T/ET at the Daman site (0.63) was higher than that at the Arou and Huyanglin sites (0.55) due to the application of plastic film mulching. The effect of leaf area index (LAI) on seasonal variations in T/ET was strong for Arou (R2 = 0.74) and Daman (R2 = 0.76) sites, but weak for Huyanglin (R2 = 0.44) site. Daily T/ET derived using the uWUE method agreed with that using the isotope and lysimeter/eddy covariance methods during the peak growth season at the Daman site. The estimated T using the uWUE method showed consistent seasonal and diurnal patterns and magnitudes with that using the sap flow method at the Huyanglin site. In addition, the uWUE method can estimate ecosystem T/ET without scaling issues, and can effectively capture T/ET variations in relation to LAI changes and the abrupt T/ET changes in response to individual irrigation events. These advantages make the uWUE method effective for ET partitioning at the ecosystem scale, and can be used for water resources management by predicting seasonal pattern of actual plant water requirements to support irrigation strategies in arid regions.