Intrinsic water-use efficiency (iWUE) in trees, defined as the ratio of photosynthetic rate (A) to stomatal conductance (g(s)), is a key indicator characterizing the carbon-water balance in trees. Previous studies have suggested that seasonal variation in iWUE is primarily controlled by g(s) in arid regions; however, the main driving factors in humid regions remain unclear. Therefore, this study utilized 8 years of high-resolution tree-ring delta C-13 and delta O-18 data from Pinus massoniana (Lamb.) (coniferous) and Sassafras tzumu (Hemsl.) (broadleaf) in the humid East Asian monsoon region to reconstruct the seasonal dynamics of iWUE. We estimated iWUE based on delta C-13 and derived the leaf water delta O-18 enrichment (Delta O-18(lw)) from delta O-18 to represent g(s), thereby distinguishing the relative contributions of A and g(s) to variations in iWUE. Both species exhibited synchronous seasonal iWUE patterns: decreasing from spring to summer before autumn recovery. Dual-isotope analysis revealed that iWUE seasonal variations were primarily driven by fluctuations in A, contrasting with g(s)-dominated mechanisms in arid regions. Summer iWUE declines resulted from reduced A, constrained by relatively low CO2 and high temperatures. This study reveals an A-dominated regulatory mechanism of iWUE in humid regions, providing a theoretical basis for more accurate predictions of forest carbon-water coupling under varying moisture conditions.