The concentration of CO is measured by tunable diode laser absorption spectroscopy (TDLAS) technology. The experimental data of measurement signals are inversely processed by applying the overall second harmonic least squares data processing algorithm. The principal sources of measurement results uncertainty are analyzed during measurement of CO concentration by TDLAS. The uncertainty components, standard uncertainty, and synthesis uncertainty caused by various factors are evaluated with the direct evaluation method. The results indicate that the major factors affecting measurement uncertainty are the uncertainty of the apparatus, concentration definite value uncertainty of calibration gas, and the uncertainty indicated by ambient temperature variation and supply voltage fluctuation.