The partitioning of evapotranspiration (ET) into soil evaporation (E) and crop transpiration (T) is fundamental for accurately monitoring agro-hydrological processes, assessing crop productivity, and optimizing water management practices. In this study, the isotope tracing technique was used to partition ET and quantify the root water uptake sources of winter wheat during the 2014 and 2015 growing seasons in Beijing, China. The correlations between seasonal ET partitioning and the leaf area index (LAI), grain yield, and water use efficiency (WUE) were investigated. The fraction of T in ET ( F T ) between the greening and harvest seasons was 0.82 on average and did not vary significantly among the different irrigation and fertilization treatments ( p > 0.05). However, the values of F T during the individual growth periods were remarkably distinct (ranging from 0.51 to 0.98) among the treatments. The seasonal variability in F T could be effectively explained via a power-law function of the LAI ( F T = 0.61 LAI 0.21 , R 2 = 0.66, p < 0.01). There was no significant relationship between F T and the grain yield or WUE ( p > 0.05). The total T during the jointing–heading and heading–filling periods (T jf ) had significantly quadratic relationships with the crop yield and WUE ( p < 0.01). Both the crop yield and the WUE had high values under the T jf range of 117.5–155.8 mm. Furthermore, the WUE was improved by increasing the ratio of E in ET ( F E ) during the greening–jointing period and by reducing F E during the filling–harvest period. Winter wheat mainly utilized soil water from the 0–20 cm (67.0 %), 20–70 cm (42.0 %), 0–20 cm (38.7 %), and 20–70 cm (34.9 %) layers during the greening–jointing, jointing–heading, heading–filling, and filling–harvest periods, respectively. This indicated that the irrigation wetting layer should be controlled at depth of 70 cm to conserve water.