<p>This study explores the effects of biochar (BC) amendments at varying levels (0%, 2%, and 5%) on the molecular and structural characteristics of humic substances (HS), specifically humic acids (HA) and fulvic acids (FA), in full-scale chicken manure compost. The results showed an increase in SUVA₂₅₄ and HIX, accompanied by a decrease in E₂/E₃ and S_R of compost HA as BC levels increased, suggesting an enhancement in the aromaticity and average molecular weight (MW) of HA. These changes are likely driven by BC-facilitated microbial activity and polymerization processes. In contrast, the aromaticity of FA remained largely unchanged. High performance size exclusion chromatography demonstrated increases in both weight-average MW (Mw) and number-average MW (Mn) of HA, indicating that BC addition enhanced the humification and stabilization of HA. Fourier-transform infrared (FTIR) spectroscopy showed that BC addition had minimal impact on the functional group composition of HA and FA, but it altered the proportions of carbon types. Electron paramagnetic resonance (EPR) analysis revealed higher levels of persistent free radicals (PFRs) in HA compared to FA, which correlated with the larger molecular size and higher degree of condensation of HA. Additionally, BC amendments reduced PFRs formation in HA while promoting them in FA, suggesting that BC additions play distinct roles in PFRs formation and their potential environmental effects. These findings highlight the potential of BC amendments to improve compost quality by promoting the formation of stable and complex humic substances with enhanced reactivity.</p>