In tunable diode laser absorption spectroscopy technology, the direct absorption method can directly obtain absorption line profile function through fitting the transmitting light intensity. But at present, the wavelength modulation method with higher signal-to-noise ratio can′t measure it effectively. A measurement theory and method of absorption line profile function based on multiple harmonics was proposed. Through the analysis of harmonic expressions, the ratio between the 2nd harmonic and 4th harmonic at the center frequency was only related to the absorption line profile function and modulation index. When the modulation index m was 2.492 8, the ratio between the 2nd harmonic and 4th harmonic was fixed to 2.186 2 no matter what the ratio of Gauss linewidth and Lorentz linewidth was. According to the characteristics of this point, the spectral line half width can be obtained firstly, and then it can be applied in 2f/1f calibration-free measurement of gas concentration. In the experiment, CO concentration has been measured using 2 326.82 nm spectral line through the proposed method. The error between the proposed method and traditional direct absorption method was less than 2%. An important theoretical basis for the measurement of absorption line profile function in wavelength modulation method has been provided by this proposed method, and the traditional theory of 2f/1f calibration-free method has been further improved.