Chlorophyll is the main light-harvesting pigment in leaves, facilitating photosynthesis and indicating the supply of nitrogen for photosynthetic enzymes. In this study, we explore the feasibility of integrating leaf chlorophyll content (Chlleaf) into a Terrestrial Biosphere Model (TBM), as a proxy for the leaf maximum carboxylation rate at 25°C (Vmax25), for the purpose of improving carbon and water flux estimation. Measurements of Chlleaf and Vmax25 were made in a deciduous forest stand at the Borden Forest Research Station in southern Ontario, Canada, where carbon and water fluxes were measured by the eddy covariance method. The use of Chlleaf-based Vmax25 in the TBM significantly reduces the bias of estimated gross primary productivity (GPP) and evapotranspiration (ET) and improves the temporal correlations between the simulated and the measured fluxes, relative to the commonly employed cases of using specified constant Vmax25, leaf area index (LAI)-based Vmax25 or specific leaf area (SLA)-based Vmax25. The biggest improvements are found in spring and fall, when the mean absolute errors (MAEs) between modelled and measured GPP are reduced from between 2.2–3.2 to 1.8gCm−2d−1 in spring and from between 2.1–2.8 to 1.8gCm−2 d−1 in fall. The MAEs in ET estimates are reduced from 0.7–0.8mmd−1 to 0.6mmd−1 in spring, but no significant improvement is noted in autumn. A two-leaf upscaling scheme is used to account for the uneven distribution of incoming solar radiation inside canopies and the associated physiological differences between leaves. We found that modelled Vmax25 in sunlit leaves is 34% larger than in the shaded leaves of the same Chlleaf, which echoes previous physiological studies on light acclimation of plants. This study represents the first case of the incorporation of chlorophyll as a proxy for Vmax25 in a two-leaf TBM at a forest stand and demonstrates the efficacy of using chlorophyll to constrain Vmax25 and reduce the uncertainties in GPP and ET simulations.