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Accurately measuring the differential molecular absorption cross section is the key to obtaining a high-precision concentration of atmospheric trace gases in a differential absorption lidar (DIAL) system. However, the CO2 absorption line is meticulous at 1.6 μm, easily translating and broadening because of the change of temperature and pressure. Hence, measuring the vertical profile of atmospheric temperature and pressure to calculate the vertical profile of the CO2 weight parameter is necessary. In general, measuring atmospheric temperature and pressure has a certain amount of uncertainty. Therefore, this study proposes the concept of a balanced on-line wavelength, where the differential molecular absorption cross section is larger and the CO2 weight parameter is insensitive to the uncertainty of atmospheric temperature and pressure. In this study, we analyzed the influence of uncertainty on the CO2 weight parameter at every preselected wavelength, as well as determined an appropriate wavelength near one of the absorption peaks. Our result shows that 1572.023 nm should be one of the appropriate balanced on-line wavelengths. The measurement errors of the mixing ratio of CO2 molecule in this wavelength are only 0.23% and 0.25% and are caused by 1 K temperature error and 1 hPa pressure error, respectively. This achievement of a balanced on-line wavelength will not only depress the requirement of the laser’s frequency stabilization but also the demand for measurement precision of the atmospheric temperature and pressure profile. Furthermore, this study can achieve the exact measurement of the vertical profile of atmospheric CO2 based on an independent differential absorption laser.