Summary Intrinsic water‐use efficiency (iWUE), a key index for carbon and water balance, has been widely estimated from tree‐ring δ¹³C at annual resolution, but rarely at high‐resolution intraseasonal scale. We estimated high‐resolution iWUE from laser‐ablation δ¹³C analysis of tree‐rings (iWUE_iso) and compared it with iWUE derived from gas exchange (iWUE_gas) and eddy covariance (iWUE_EC) data for two Pinus sylvestris forests from 2002 to 2019. By carefully timing iWUE_iso via modeled tree‐ring growth, iWUE_iso aligned well with iWUE_gas and iWUE_EC at intraseasonal scale. However, year‐to‐year patterns of iWUE_gas, iWUE_iso, and iWUE_EC were different, possibly due to distinct environmental drivers on iWUE across leaf, tree, and ecosystem scales. We quantified the modification of iWUE_iso by postphotosynthetic δ¹³C enrichment from leaf sucrose to tree rings and by nonexplicit inclusion of mesophyll and photorespiration terms in photosynthetic discrimination model, which resulted in overestimation of iWUE_iso by up to 11% and 14%, respectively. We thus extended the application of tree‐ring δ¹³C for iWUE estimates to high‐resolution intraseasonal scale. The comparison of iWUE_gas, iWUE_iso, and iWUE_EC provides important insights into physiological acclimation of trees across leaf, tree, and ecosystem scales under climate change and improves the upscaling of ecological models.