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Interaction between soil aggregates and humic substances directly determine soil carbon (C) sequestration and stabilization dynamics. This study investigated the influence of different tillage systems on soil humus composition and humic acid (HA) structural characteristics in water stable soil aggregates in the 0−20 and 20−40 cm soil depths. Tillage systems included no-tillage (NTS) and conventional tillage (CTS) both with corn straw returned, and conventional tillage without corn straw returned (CT). Fluorescence, Fourier transform infrared (FTIR) and 13C nuclear magnetic resonance (13C NMR) spectroscopies were employed to comprehensively characterize soil HA structure in soil aggregate fractions. The results showed that large quantities of humus C was stored in the large macro-aggregates (> 2 mm) and silt/clay ( 2 mm aggregates were dominated by aliphatic, methoxyl, hydrophobic and carboxyl C groups, and this result was more evident in NTS indicating improved soil C stability. Comparing all tillage systems, NTS enhanced aliphatic, methoxyl and hydrophobic C in > 2 mm aggregates, which indicate improved soil C stability. The 13C NMR results of bulk soil demonstrated that NTS enhanced alkyl, O-alkyl and methoxyl/N-alkyl C in the 0−20 cm. We therefore concluded that NTS is the better system to effectively improve soil C sequestration and stability.