Gas exchange, online isotope discrimination, and chlorophyll fluorescence measurements were conducted on fully expanded leaves of grey alder (Alnus incana) and holm oak (Quercus ilex) tree saplings under different fractions of blue light (0–100%). Blue light was supplemented with red light to maintain a constant total light intensity. A detailed description of the measurements is provided in Diao et al. (2025) (https://doi.org/10.1093/jxb/eraf305). These measurements generated a dataset describing the responses of key leaf physiological parameters to varying blue/red light fractions, including net photosynthetic rate, stomatal conductance, mesophyll conductance, the maximum carboxylation rate of RuBP, non-photochemical quenching, etc. The dataset was further used for photosynthetic limitation analysis to disentangle the relative biochemical, mesophyll, and stomatal limitations on photosynthesis under different light-quality conditions. The results of the photosynthetic limitation analysis are also provided here.