Biochar is a promising carbon sequestration strategy, however, the mechanisms underlying the regulation of microbial-derived carbon (M-C) and plant-derived carbon (P-C) in soil organic carbon (SOC) formation and stabilisation remain elusive, constraining accurate predictions of the organic carbon pool. This study examined the soil biotic and abiotic factors that influence the plant and microbial biomarkers in SOC accumulation. A 5-year field experiment was conducted in a temperate wheat-maize agroecosystem in north-western China, with three treatments: (i) no straw incorporation (C), (ii) straw incorporation (S), and (iii) straw incorporation + biochar (SB). The results showed that M-C reached the microbial carrying capacity gradually, whereas P-C was selectively and continuously accumulated, displaying a complementary S-curve pattern. Straw incorporation increased SOC, microbial biomass carbon (MBC), and dissolved organic carbon (DOC) contents, which stimulated microbial richness and enzyme activities, resulting in a 29.1 % and 25.5 % increase in M-C and P-C in SOC, respectively. The stimulated SOC mineralisation (26.2 %) led to significantly lower SOC content in S compared to the SB practice. Biochar combined with straw decreased DOC content (18.5 %) in comparison with straw incorporation, which suppressed microbial and enzyme activities, particularly in Actinobacteriota (12.3 %) and β-N-acetyl-glucosaminidase (24.2 %). It resulted in a 10.9 % and 14.3 % increase in M-C and fungal-to-bacterial necromass carbon ratio (F/B), respectively, while decreasing P-C by 9.6 % over the 5 years. Overall, straw incorporation with biochar effectively enhanced M-C in SOC and reduced SOC mineralisation, suggesting its potential to augment the quantity and stability of SOC pools and mitigate global climate change.