AbstractWhile high soil carbon stability had been well known for biochar‐amended soils, how conversion of crop residues into biochar and subsequent biochar amendment (BA) would favor microbial carbon use and carbon sequestration had not been clearly understood. In this study, topsoil samples were collected from an upland soil and a paddy soil, both previously amended with straw and straw‐derived biochar. These samples were incubated with 13C‐labeled maize residue (LMR) for 140 days to compare carbon mineralization, metabolic quotient (qCO2), and microbial carbon use efficiency (CUE) under laboratory incubation. 13C‐phospholipid fatty acid (13C‐PLFA) was used to trace the use of substrate carbon by soil microorganisms. Comparing to straw amendment (SA), BA significantly decreased the native soil organic carbon (SOC) mineralization rates by 19.7%–20.1% and 9.2%–12.0% in the upland and paddy soils, respectively. Meanwhile, total carbon mineralization from the newly added LMR was significantly decreased by 12.9% and 11.1% in the biochar‐amended soils, compared with the straw‐amended soils from the upland and paddy sites, respectively. Furthermore, compared to non‐amended soils, the qCO2 value was unchanged in straw‐amended soils, but was notably decreased by 15.2%–18.6% and 8.9%–12.5% in biochar‐amended upland and paddy soils, respectively. Microbial CUE was significantly greater in biochar‐amended soils than in straw‐amended soils due to the increasing dominance of fungi in carbon utilization. Compared to SA, BA increased CUE by 23.0% in the upland soil and 21.2% in the paddy soil. This study suggests that BA could outperform SA in the long term to enhance the biological carbon sequestration potential of both upland and paddy soils. This could be due mainly to biochar input as a special substrate to promote microbial community evolution and increase the fungal utilization of carbon substrates, especially for the soil with lower SOC levels.