The performance of a home-built tunable diode laser (TDL) spectrometer has been optimized regarding multi-line detection of carbon dioxide in natural gases. In the regime of the (3001)III;←(000) band of 12CO2 around 1.6 μm, the dominating isotope species 12CO2, 13CO2, and12C18O16O were detected simultaneously. In contrast to most established techniques, selective measurements are performed without any sample preparation. This is possible since the CO2 detection is free of interference from water, ubiquitous in natural gases. Detection limits in the range of a few ppmv were obtained for each species utilizing wavelength modulation (WM) spectroscopy with balanced detection in a long-path absorption cell set-up. Linear calibration plots cover a dynamic range of four orders of magnitude, allowing for quantitative CO2 detection in various samples, like soil and breath gas. High isotopic resolution enables the excellent selectivity, sensitivity, and stability of the chosen analytical concept. The obtained isotopic resolution of typically ± 1.0 % and ± 1.5 % (for 3 vol. % and 0.7 vol. % of CO2, respectively) offers a promising analytical tool for isotope-ratio determination of carbon dioxide in soil gas. Preliminary experiments on soil respiration for the first time combine the on-line quantification of the overall carbon dioxide content with an optode sensor and isotopic determination (TDL system) of natural gas species.