This study explored the effects of different proportions of organic and inorganic fertilizers on soil humus characteristics based on the 16-year long-term localization experiment. The humic acid (HA) and fulvic acid (FA) components in the humus are separated and purified, and studied the changes of HA and FA contents and structure utilized chemical analysis and various spectroscopy techniques under three fertilization of F1 (70% chemical fertilizer +30%organic fertilizer), F2 (50% organic fertilizer+50% chemical fertilizer) and F3 (100% organic fertilizer). The results showed that the effect of F3 on improving HA and FA content was better than that of F1 and F2 treatments, indicating that the higher application rate of organic fertilizer, the higher content of soil humus components. Elemental analysis showed that different ratios of organic and inorganic fertilizers had different effects on the content and atomic ratio of HA and FA. F2 and F3 both increase the degree of HA condensation, reduce the degree of oxidation and polarity, and the effect of F3 is obvious; the application of organic fertilizer can also promote the formation of nitrogen compounds in HA, and the effect is the best in F2; The application of organic fertilizer also increased the degree of condensation of FA, reduced the degree of oxidation and polarity, and promoted the formation of nitrogen-containing compounds, especially in the F2 treatment. Infrared spectroscopy analysis of humus showed that compared with F1, F2 and F3 increased the content of aliphatic compounds and carbohydrates in HA, and the carboxyl group and fat group content of FA also increased, and it was the most significant in F3 treatment. F2 treatment reduced the content of fatty hydrocarbons in HA, and the carbohydrates in FA reached the highest. ¹³C NMR spectroscopy analysis showed that the application of organic fertilizer increased the degree of aromatization of HA and FA and decreased the degree of aromatization in the three treatments, and the change of HA functional group in F2 treatment was significant, FA changed more obvious in F3 treatment. In summary, organic fertilizer significantly increased the content of soil humus components and increased the degree of aromatization of HA and FA, but the formation mechanism of HA and FA under different amounts of organic fertilizer was different.