Carbon sequestration by application of organic materials and biochar in soil is an important strategy to increase soil organic carbon (SOC), but the stability of SOC, particularly humic substances (HS) vary with the types of organic material. Cotton straw and its derived compost and biochar were added with equivalent carbon content to soil and incubated for 180 days. The structural characteristics of humic acid (HA), fulvic acid (FA) and humin (Hu) were investigated using solid-state ¹³C nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. The results showed that biochar treatment increased the aryl C of HA, FA, and Hu by 1.38%, 1.68%, and 10.46% compared to straw treatment and increased the aryl C of HA, FA, and Hu by 1.46%, 1.99% and 2.01% compared to compost treatment. The O-alkyl C of HA was 10.59% and 10.65% in high biochar/straw and biochar/compost ratios respectively, while it was 9.81% and 9.61% in low biochar/straw and biochar/compost ratios. In addition, the O-alkyl C of FA was 62.83% and 58.48% in high ratios of biochar/straw and biochar/compost, respectively, while it was 55.85% and 55.94% in low ratios of biochar/straw and biochar/compost. These results suggest that biochar is advantageous for aryl C formation of FA and Hu due to its high aryl C content, whereas straw or compost is advantageous for alkyl C formation of HA. The stability of aryl C and O-alkyl C of HA, FA, and Hu can be improved in soils by incorporating biochar in combination with straw or compost.