Biochar is recognized for its role in carbon sequestration and emission mitigation in farmland topsoil. However, the mechanisms by which biochar affects soil organic carbon (SOC), its composition, and stability, in the topsoil (0-20 cm) and subsoil (140-160 cm) remain unclear. Applying biochar to the calcareous farmland topsoil significantly increased the topsoil SOC contents by 33 % after a decade, with a 5 % increase in dissolved organic carbon (DOC) contents (topsoil) and a substantial increase of 162 % in subsoil DOC contents. Additionally, humic substances showed an increase of 24 % (topsoil), while low-molecular-weight water-extracted DOC exhibited a remarkable increase of 142 % in the subsoil. The application of biochar significantly increases the contents of SOC, DOC, and microbial biomass carbon (MBC) in the topsoil, as well as SOC and DOC contents in the subsoil. However, a slight decrease is observed for MBC content in the subsoil. Biochar-amended soil significantly sup-pressed enzyme activity in the topsoil and decreased alpha diversity in topsoil and subsoil while increasing the content of mineral-associated soil organic matter (MAOM). These observed changes are conducive to stabilizing SOC, emphasizing MAOM formation as a primary mechanism for carbon sequestration in both topsoil and subsoils. This study provides evidence that biochar contributes to the long-term organic carbon sequestration in calcareous farmland, highlighting the importance of considering both topsoil and subsoil when evaluating the dynamic impacts of biochar on SOC.