Natural gas hydrates, as a clean and green energy source, are widely distributed in extreme environments near the seabed. They release relatively low concentrations of carbon dioxide gas dissolved in water. Therefore, precise detection of changes in the concentration of dissolved carbon dioxide gas in water is an effective means to accurately characterize the distribution of natural gas hydrates. Compared to direct absorption spectroscopy and near-infrared spectroscopy systems, the tunable diode laser absorption spectroscopy-wavelength modulation spectroscopy (TDLAS-WMS) system, which operates in the mid-infrared region, can effectively improve the detection limit performance of the system. However, due to the complex spectral lines in the mid-infrared region, it is also more susceptible to overlapping interference from adjacent spectral lines. In order to improve the measurement accuracy of the TDLAS-WMS system in complex spectral lines region, we proposed a spectral line separation method and applied it to the separation of overlapping second harmonic spectral lines with similar absorption strength. The influence coefficients calculated by the spectral line separation method are combined with the system calibration process to achieve real-time separation. The method was tested for separation effectiveness in combination with a single-axis mid-infrared carbon dioxide sensing system. The relative error was less than 1.205% for the measured concentration. The minimum detection limit of the system was 0.277 ppmv. This method improved the accuracy in complex spectral line regions while maintaining high stability and high sensitivity performance.