Soil organic matter is an important part of soil , and plays an important role in soil fertility, environmental protection and sustainable development of agriculture .As the main body of soil organic matter, humus has an effect on a series of properties and forms of soil. Its quantity, composition and properties can reflect certain conditions and processes of soil formation, and it is an important index of soil fertility. Humus is a complex mixture of species, and there is no definite molecular weight and only molecular weight distribution. Its molecular composition and chemical structure are still not clear until now, which largely limits the further research of humic acid. Due to the uncertainty of the composition of the humus molecules, there are some limitations of various methods. Optimizing the search for a more accurate and reliable humic acid characterization method has become a hot spot in the current research. Fertilization modifies the composition and structure of humic acid in soil, but the short-term influence degree is difficult to be detected by conventional methods. This paper uses 38 years of long-term black soil positioning test, through the separation and purification of humic component HA, thermal properties analysis, fourier transform infrared spectroscopy, 13C nuclear magnetic resonance spectroscopy and fluorescence spectroscopy analysis technology. The effects of organic fertilizer and inorganic fertilizer on the molecular structure of HA in black soil were analyzed from the point of view of material structure. The results showed that the mechanism of organic fertilizers application (MNPK) and CK (no fertilization) to increase crop yields was that MNPK fertilization increased soil organic carbon content compared with CK, at the same time, the HA content of humus could be extracted from soil, and the content of aliphatic C in soil HA increased and the structure was simplified. The lipids in soil organic matter were mainly fat, wax and resin, which had a significant influence on the decomposition of soil organic matter, thus affecting nutrient release and plant growth, and the increase of soil nutrients promoted the increase of maize yield under MNPK fertilization. The analysis showed that the total reaction heat of MNPK fertilization treatment soil HA, 2 920/1 720 and 2 920/2 850 value was higher than CK, and 13C nuclear magnetic resonance spectroscopy showed that the molecular structure of HA in black soil was obviously aliphatic after MNPK fertilization, and increased the soil aliphatic C content of HA, reduced the aromatic C content. Fluorescence emission spectra showed that the f450/500 value of MNPK-treated HA increased significantly compared with the control, indicating that the degree of aromatization of soil HA decreased with the application of organic fertilizer. The above analysis results showed that: after the application of organic and inorganic fertilizers, the molecular structure of HA in the black soil became aliphatic and simplistic; the combination of multiple spectroscopic techniques could mutually verify the accuracy of the results. At the same time, the test results also proved that once organic fertilizer was applied in the rotation period, the soil organic carbon and the Aliphatic C content of soil HA could be increased, and the crop yield and soil fertility could be increased.