【Objective】Contributions of long-term fertilization to soil fertility and carbon sequestration mechanism of aggregates in dark brown soil were explored. 【Method】A long-term field experiment was established to study effects of fertilization on contents and composition of humic substabce—humic acid (HA) in aggregates of dark brown soil, using element analysis and Fourier transformed infrared (FTIR) spectra. The experiment was designed to have four treatments in fertilization regime, i.e. no fertilizer (CK); horse manure (M) (once 3 year−1); low-level chemical nitrogen and phosphorus fertilizers (N1P1); and horse manure combined with high-level chemical nitrogen and phosphorus fertilizer (M+N2P2). Soil samples collected from the treatments were wet-sieved for macroaggregates (>2 mm and 2~0.25 mm), microaggregates (0.25~0.053 mm) and silt/clay fraction (<0.053 mm). 【Result】The soil aggregates in the dark brown soil were composed mainly of microaggregates, which were the lowest in content of soil organic carbon (SOC) and HA, while macroaggregates (>2 mm) the highest. Long-term application of horse manure significantly increased the proportion of macroaggregates (2~0.25 mm) or by 49.06% in Treatment M, but decreased by 31.04% in Treatment M+N2P2. Treatment N1P1 did not have much effect on aggregates composition. Application of either horse manure or chemical fertilizers increased SOC content, but did not have much impact on content of HA. In Treatment CK, HA in macroaggregates (2~0.25 mm) was quite lower in condensation degree, but the highest in oxidation degree and aliphaticity, while HA in microaggregates the highest in condensation degree, aromaticity, “age” and stability. Compared with Treatment CK, all the other treatments (i.e., M, N1P1, M+N2P2) reduced the condensation degree, increased the contents of aliphatic hydrocarbon carbon (C) and aromatic-C and enhanced aromaticity as the result of decreased ratio of aliphatic hydrocarbon-C to aromatic-C (aliphatic hydrocarbon-C/aromatic-C ratio) in the HA in macroaggregates, while reducing the condensation degree and increasing the contents of aliphatic hydrocarbon-C, and aromatic-C, and the ratio of aliphatic hydrocarbon-C/aromatic-C , and hence aliphaticity of the HA in microaggregates. In the experiment field, molecular structure of HA of silt/clay fractions varied with the treatment. Compared with CK, Treatment M increased aliphatic hydrocarbon-C and reduced aromatic-C in content and as a result, increased aliphatic hydrocarbon-C/aromatic-C ratio, condensation degree and aliphaticity of the HA. In Treatment M+N2P2, the HA in aggregates of silt/clay fractions increased in condensation degree and aromaticity as the result of increased content of aromatic-C and decreased aliphatic hydrocarbon-C/aromatic-C ratio. Moreover in Treatment N1P1, the HA in aggregates of silt/clay fractions decreased in condensation degree, aliphatic hydrocarbon-C and aromatic-C contents and aliphatic hydrocarbon-C/aromatic-C ratio, and as a result, increased in aromaticity. 【Conclusion】 Long-term application of horse manure increases the content of SOC, promotes formation of 2~0.25 mm macroaggregates, contributes favorably to stabilization of the HA in macroaggregates, thus helping carbon sequestration. In addition, it also enhances activity of the HA in microaggregates (0.25~0.053 mm) and aggregates of silt/clay fractions (<0.053 mm), which contributes to soil fertility supply. These aggregates can be used as important indicators for evaluating effects of long-term application of horse manure on quality of humic substance in the soil.