Elevated CO₂ generally increases wheat grain yield, which may be affected by nitrogen fertilization. The aim of this study was to investigate the responses of carbohydrate metabolism enzymes in source leaves and sink spikes in wheat to elevated CO₂ and nitrogen fertilization and their relation to grain yield. The wheat plants were grown at different atmospheric [CO₂] (400 and 800 ppm) and nitrogen fertilizer rates, and the activities of 13 key carbohydrate metabolism enzymes, carbon and nitrogen concentrations in leaves and spikes, and grain yield were analyzed. Elevated CO₂ (e[CO₂]) increased the photosynthetic rate while decreased the chlorophyll content in flag leaf, compared with plants grown at ambient CO₂ (a[CO₂]). In source leaf, activity of invertase, including vacInv and cwInv, were depressed by e[CO₂]. In leaf the PGI activity was enhanced, while the Ald activity was reduced by high N, compared with normal N supply. Among the enzymes involved in starch biosynthesis, only PGM activity was enhanced by e[CO₂] in spike, compared with those grown at a[CO₂]. For the glycolysis pathway in spike, the PFK activity was not affected by e[CO₂], but the Ald activity was promoted by e[CO₂], in relation to those grown at a[CO₂]. Grain yield and shoot biomass were significantly enhanced by e[CO₂], compared with a[CO₂]. In addition, high N supply enhanced the grain number per spike in wheat grown under e[CO₂]. The activities of major carbohydrate metabolism enzymes in source and sink organs show different responses to e[CO₂] and N fertilizer rates, which could have contributed to the changes in grain yield. The variation in N supply could be a key contributing factor to the variable responses of wheat grain yield to e[CO₂].