Abstract: Stable isotope technique has been used to attribute carbon dioxide (CO₂) emissions to different processes, and over the past two decades their use has come to the forefront as a promising tool for CO₂ source partitioning. Accurate quantification of carbon dioxide (CO₂) (and other greenhouse gases) as a result of the activities of soil microorganisms is important for understanding soil respiration process and predicting carbon exchange between soil and atmosphere. Recently, laser-based analyzers have become commercially available for simultaneously measurements of the concentrations and the isotopic composition of CO₂, using cavity-enhanced absorption spectroscopy for the analysis of ambient air samples. However, despite this innovation for the possibility of in situ field measurements, their use raises unique demanding for an on-line gas sampling and displacement auxiliary. In order to realize the continuous measurement of soil microbial respiration, this study aimed to establish an online analytical method suitable for continuous in situ monitoring of CO₂ concentration and its δ¹³C value from soil microbial respiration gases. Based on CRDS technology, a self-developed multi-channel online gas exchange and sample introduction device was integrated with a carbon stable isotope analyzer (G2201-i) to construct an automated continuous monitoring system. Tests using a series of standard gases demonstrated that the system achieved high accuracy and precision in measuring CO₂ concentration and δ¹³C values. The measured values were consistent with reference values, with a coefficient of variation for concentration measurements less than 0.1% and δ¹³C measurements below 0.5‰, meeting practical testing requirements. The application of this system to in situ soil microbial respiration studies yielded results consistent with those obtained by traditional gas chromatography-isotope ratio mass spectrometry (GC-IRMS) (correlation coefficients R² were 0.999 and 0.997, and slopes were 0.998 and 0.995, respectively), validating the reliability of the method. The established method enables synchronous, high-precision, and automated in situ measurement of CO₂ and its isotopic composition from soil microbial respiration. It exhibits excellent timeliness and stability, providing a key technical solution for in-depth investigation into soil organic carbon transformation processes and microbial driving mechanisms. This method holds significant application value for the precise quantification of carbon fluxes in terrestrial ecosystems.