To investigate the effects of biochars on humus composition and water-stable aggregate characteristics in the tobacco-planting black soil region of Northeast China, three treatments including low quantity of biochar(5 t/hm²,C1), high quantity of biochar (25t/hm², C2) and no biochar application (CK), were setup in 2019 to 2020 in the field, the composition of soil humus and the distribution of water-stable aggregates were analyzed, and humic acid chemical structures were determined with Fourier transform infrared spectroscopy (FTIR) and ¹³C nuclear magnetic resonance spectroscopy (¹³C-NMR). The results showed that C1 and C2 reduced fulvic acid by 16.90% and 40.85%, significantly increased the content of humic acid by 14.86% and 33.78%, respectively, the proportion of humic acid in soil humus also significantly increased. FTIR and ¹³C-NMR analyses showed that C2 decreased the I₂₉₂₀/I₁₆₂₀ absorption peak intensity ratio of soil humic acid and the ratio of aliphatic carbon/aromatic carbon by 11.82% and 13.04%, respectively, suggesting the application of high quantity of biochar could enhance the aromatization degree of humic acid and reduce the proportion of aliphatic structure. The addition of biochars increased the proportion of soil large aggregates (>0.25mm), and C2 had the most significant effect. The content of humic acid was significantly positive correlated with the content of 2 ~ 0.25 mm aggregates, and the characteristics of aliphatic functional groups of humic acid molecules were significantly positive correlated with >2 mm size aggregates. C1 and C2 also significantly increased the yield of tobacco leaves. In conclusion, biochar can improve the content and structure of humic acid in soil humus, which is conducive to the formation of soil aggregates, improve the potential of soil carbon sequestration, and also have a positive impact on the crop yield.