Abstract: Tunnel engineering significantly impacts soil structure and function. Soil microorganisms and plants, being critical sources of the soil organic carbon pool, play a central role in the global carbon cycle. The ecological environment of the Qinghai-Tibet Plateau is fragile and susceptible to anthropogenic disturbances, which can affect the soil carbon pool and subsequently influence global climate change. To investigate the impact of large-scale engineering on the sources of soil organic carbon on the Qinghai-Tibet Plateau, this study focuses on a specific tunnel on the plateau. It compares affected and non-affected areas across different elevations (3240 m, 3420 m, and 3600 m) and soil depths (0 - 10 cm and 10 - 30 cm). Using amino sugars and lignin as biomarkers, the research characterizes the microbial and plant sources of organic carbon in soil aggregates. The study aims to explore the influence of tunnel construction on the sources of organic carbon in particulate (> 0.053 mm) and mineral-associated (< 0.053 mm) soil aggregates. The results showed that tunnel construction had no significant effects on the contents of glucosamine, galactosamine, total amino sugars, fungi and total lignin phenols in soil aggregates (P > 0.05), but significantly reduced the contents of muramic acid, mannosamine, and bacterial residue carbon in soil aggregates. There was no significant difference in amino sugars in soil particulate aggregates across different altitudes at the 0-10 cm soil depth. However, at the 10-30 cm soil depth, amino sugar content at 3420 m was significantly higher than that at 3240 m and 3600 m. For mineral-bound aggregates, amino sugar content at 3420 m were lower than those at the other altitudes. Meanwhile, in the mineral-bound aggregates of the tunnel-affected area, the average contributions of microbial residue carbon and lignin phenols to aggregate organic carbon were 26.36% and20.65% respectively, which were significantly lower than those in the control area (P < 0.05).The study indicated that tunnel construction reduces the stability of soil aggregates, especially that of mineral-bound aggregates. Specifically, under the interactive effects of tunnel construction, altitude and soil depth, the microbial residue carbon content of mineral-bound aggregates and their contribution rate to soil organic carbon decrease significantly, whereas the microbial and plant-derived carbon contributions to organic carbon in soil particulate aggregates are not significantly affected by tunnel construction.