The O2 : CO2 exchange ratio (ER) between terrestrial ecosystems and the atmosphere is a key parameter for partitioning global ocean and land carbon fluxes. The long-term terrestrial ER is considered to be close to 1.10 mol of O2 consumed per mole of CO2 produced. Due to the technical challenge in measuring directly the ER of entire terrestrial ecosystems (EReco), little is known about variations in ER at hourly and seasonal scales, as well as how different components contribute to EReco. In this modeling study, we explored the variability in and drivers of EReco and evaluated the hypothetical uncertainty in determining ecosystem O2 fluxes based on current instrument precision. We adapted the one-dimensional, multilayer atmosphere–biosphere gas exchange model “CANVEG” to simulate hourly EReco from modeled O2 and CO2 fluxes in a temperate beech forest in Germany. We found that the modeled annual mean EReco ranged from 1.06 to 1.12 mol mol−1 within the 5-year study period. Hourly EReco showed strong variations over diel and seasonal cycles and within the vertical canopy profile. The determination of ER from O2 and CO2 mole fractions in air above and within the canopy (ERconc) varied between 1.115 and 1.15 mol mol−1. CANVEG simulations also indicated that ecosystem O2 fluxes could be derived with the flux-gradient method using measured vertical gradients in scalar properties, as well as fluxes of CO2, sensible heat and latent energy derived from eddy covariance measurements. Owing to measurement uncertainties, however, the uncertainty in estimated O2 fluxes derived with the flux-gradient approach could be as high as 15 µmol m−2 s−1, which represented the 90 % quantile of the uncertainty in hourly data with a high-accuracy instrument. We also demonstrated that O2 fluxes can be used to partition net CO2 exchange fluxes into their component fluxes of photosynthesis and respiration if EReco is known. The uncertainty of the partitioned gross assimilation ranged from 1.43 to 4.88 µmol m−2 s−1 assuming a measurement uncertainty of 0.1 or 2.5 µmol m−2 s−1 for net ecosystem CO2 exchange and from 0.1 to 15 µmol m−2 s−1 for net ecosystem O2 exchange, respectively. Our analysis suggests that O2 measurements at ecosystem scale have the potential to partition net CO2 fluxes into their component fluxes, but further improvement in instrument precision is needed.