The isotopic compositions of water fluxes provide valuable insights into the hydrological cycle and are widely used to quantify biosphere-atmosphere exchange processes. However, the combination of water isotope approaches with water flux components remains challenging. The Iso-SPAC (coupled heat, water with isotopic tracer in soil-plant-atmosphere-continuum) model is a useful framework for simulating the dynamics of water flux and its components, and for coupling with isotopic fractionation and mixing processes. Here, we traced the isotopic fractionation processes with separate soil evaporation (Ev) and transpiration (Tr), as well as their mixing in evapotranspiration (E) for simulating diurnal variations of isotope compositions in E flux (δ E ). Three sub modules, namely isotopic steady state (ISS), non-steady-state (NSS), and NSS Péclet, were tested to determine the true value for the isotope compositions of plant transpiration (δ Tr ) and δ E . In situ measurements of isotopic water vapor with the Keeling-plot approach for δ E and robust eddy covariance data for E agreed with the model output (R 2 = 0.52 and 0.98, RMSD = 2.72% , and 39 W m −2 ), illustrating the robustness of the Iso-SPAC model. The results illustrate that NSS is a better approximation for estimating diurnal variations in δ Tr and δ E , specifically during the alternating periods of day and night. Leaf stomata conductance regulated by solar radiation controlled the diurnal variations in transpiration fraction (Tr/E). The study emphasized that transpiration and evaporation, respectively, acted to increase and decrease the δ 18 O of water vapor that was affected by the diurnal trade-off between them. of the land surface process of climate models [14]. The Soil-Plant-Atmosphere-Continuum (SPAC) concept is used to link to climate models to more accurately describe how soil, vegetation, and water surfaces exchange fluxes with the atmosphere. The SPAC model, which can estimate and predict evaporation and transpiration fluxes separately [15,16], have been widely used in various ecosystems to estimate and partition E flux [17][18][19]. The isotope method provides an effective tool to partition E into Tr and Ev. In particular, SPAC models involving isotopic tracers (i.e., Iso-SPAC models) are expected to be more reliable for this purpose [20][21][22]. This kind of model has the advantage of allowing assessment both by water flux (i.e., E, Tr, and Ev) and isotope composition in evapotranspiration, plant transpiration and soil evaporation (δ E , δ Tr , and δ Ev ), which expected to be more reliable for partitioning the E flux [23][24][25][26][27]. However, some previous studies have questioned the isotopic steady state (ISS), which assumes that the δ 18 O and δD of transpiration flux (δ Tr ) are operationally defined as being equal to plant-stem xylem water (δ x ) [28][29][30]. There are several models of varying complexity, including isotopic non-steady state (NSS) [31] and the non-steady state model with considering isotopic advection and diffusion (NS...