The differences of molecular characteristic of humic acid (HA) under long-term application of chemical fertilizer and manure for 34 years were determined by 13C cross polarization magic angle spinning nuclear magnetic resonance (13C CPMAS NMR) spectroscopy in order to investigate the carbon (C) sequestration in soils. The results showed that the relative content of alkyl C increased in manure treatments, compared to control and chemical fertilizer treatments. There were positive correlations between alkyl C and hydrogen to carbon (H/C) ratio (R2 = 0.84, P < 0.01), alkyl C and amounts of HA (R2 = 0.57, P < 0.01) as well as alkyl C and E4/E6 ratio (R2 = 0.55, P < 0.01). Conversely, negative correlations were found between aromatic C and alkyl C (R2 = 0.76, P = 0.01), H/C ratio and aromaticity (R2 = 0.37, P = 0.04), aromaticity and amounts of HA (R2 = 0.71, P < 0.01) as well as aromaticity and E4/E6 ratio (R2 = 0.65, P < 0.01). Compared to control treatment, the amorphous C/crystalline C ratio increased for 0.02–0.14 and 0.30–0.78 in chemical fertilizer treatments and manure treatments, separately. Moreover, the short-chain (CH2)n/Long-chain (CH2)n ratio increased for 0.03–0.37 and 0.03–0.18 in chemical fertilizer treatments and manure treatments, separately. These findings suggest that application of manure favours alkyl C sequestration in HA and tends to be more closely linked to amorphous C than crystalline C, while incorporates less short-chain (CH2)n, compared to chemical fertilizer treatments. Moreover, the E4/E6 ratio of HA increased with more alkyl C.