Terrestrial ecosystems have been serving as a strong carbon sink that offsets one-quarter of anthropogenic CO2 emissions. Carbon use efficiency (CUE), the percentage of photosynthesized carbon that is available for biomass production and other secondary carbon products, is one factor determining the carbon sink size. The global variation in CUE remains unclear, however, as recent reports disagree over the responses of CUE to temperature, dryness, forest types and stand age, and there are limited direct observations to constrain the related uncertainty. Here, we propose to infer CUE from spatially distributed observations of land–atmosphere CO2 exchange from global eddy covariance sites based on the degree of ecosystem respiration–photosynthesis coupling. Across 2,737 site-years, CUE derived from eddy covariance observations is 0.43 ± 0.12, consistent with previous inventory-based estimates (0.47 ± 0.12, n = 301) but with a better representation of spatial–temporal variation in CUE. We find that CUE consistently decreases with temperature, precipitation, light availability and stand age, with a substantial difference in the baseline CUE among biomes. Importantly, CUE of deciduous forests is typically 15% higher than that of evergreen forests, suggesting that over the long-term deciduous forests are more efficient in using photosynthate. Our study advances the understanding of the global variation in CUE and provides insights to guide best practices of forest conservation, management and restoration for carbon sequestration.