Recent advances in laser spectroscopy have allowed for real-time measurements of the 13C/12C isotopic ratio in CO2, thereby providing new ways to investigate carbon cycling in natural ecosystems. In this study, we combined an Aerodyne quantum cascade laser spectrometer for CO2 isotopes with a LI-COR LI-8100A/8150 automated chamber system to measure the δ13C of CO2 during automated closed-chamber measurements. The isotopic composition of the CO2 flux was determined for each chamber measurement by applying the Keeling plot method. We found that the δ13C measured by the laser spectrometer was influenced by water vapour and CO2 concentration of the sample air and we developed a method to correct for these effects to yield accurate measurements of δ13C. Overall, correcting for the CO2 concentration increased the δ13C determined from the Keeling plots by 3.4‰ compared to 2.1‰ for the water vapour correction. We used the combined system to measure δ13C of the CO2 fluxes automatically every two hours from intact soil, trenched soil, tree stems and coarse roots during a two-month campaign in a Danish beech forest. The mean δ13C was −29.8 ± 0.32‰ for the intact soil plots, which was similar to the mean δ13C of −29.8 ± 1.2‰ for the trenched soil plots. The lowest δ13C was found for the root plots with a mean of −32.6 ± 0.78‰. The mean δ13C of the stems was −30.2 ± 0.74‰, similar to the mean δ13C of the soil plots. In conclusion, the study showed the potential of using a quantum cascade laser spectrometer to measure δ13C of CO2 during automated closed-chamber measurements, thereby allowing for measurements of isotopic ecosystem CO2 fluxes at a high temporal resolution. It also highlighted the importance of proper correction for cross-sensitivity with water vapour and CO2 concentration of the sample air to get accurate measurements of δ13C.