Understanding the fate of organic carbon in thawed permafrost is crucial for predicting climate feedback. While minerals and microbial necromass are known to play crucial roles in the long-term stability of organic carbon in subsoils, their exact influence on carbon persistence in Arctic permafrost remains uncertain. Our study, combining radiocarbon dating and biomarker analyses, showed that soil organic carbon in Alaskan permafrost had millennial-scale radiocarbon ages and contained only 10%-15% microbial necromass carbon, significantly lower than the global average of ~30%-60%. This ancient carbon exhibited a weak correlation with reactive minerals but a stronger correlation with mineral weathering (reactive iron to total iron ratio). Peroxidase activity displayed a high correlation coefficient (p < 10-6) with Δ14C and δ13C, indicating its strong predictive power for carbon persistence. Further, a positive correlation between peroxidase activity and polysaccharides indicates that increased peroxidase activity may promote the protection of plant residues, potentially by fostering the formation of mineral-organic associations. This protective role of mineral surfaces on biopolymers was further supported by examining 1451 synchrotron radiation infrared spectra from soil aggregates, which revealed a strong correlation between mineral OH groups and organic functional groups at the submicron scale. An incubation experiment revealed that increased moisture contents, particularly within the 0%-40% range, significantly elevated peroxidase activity, suggesting that ancient carbon in permafrost soils is vulnerable to moisture-induced destabilization. Collectively, this study offers mechanistic insights into the persistence of carbon in thawed permafrost soils, essential for refining permafrost carbon-climate feedbacks.