Anaerobic microbial oxidation of hydrocarbons is a key biogeochemical process influencing hydrocarbon compositions and the carbon cycle in subsurface systems. In this study, the microbial oxidation of propane within the Chang 7 shale of the Ordos Basin was investigated through intramolecular carbon isotope analysis of thermally desorbed gases. The results show that samples affected by microbial activity exhibit elevated Δ_C-T values (3.9‰–7.9‰), δ¹³C_central values ranging from −27.2‰ to −22.9‰, and δ¹³C_terminal values from −31.1‰ to −30.5‰, significantly higher than non-degraded samples. These isotopic characteristics indicate that microorganisms preferentially oxidize the central carbon atom in propane, while the terminal carbon retains its original isotopic signature. The distinct position-specific isotopic patterns serve as sensitive indicators of microbial degradation in shale reservoirs. Furthermore, sedimentological data suggest that microbial degradation potential is closely linked to the sand supply from different sedimentary facies. Toward deeper lacustrine settings, reductions in sandy interlayer thickness and grain size, along with enhanced nanoconfinement effects, restrict nutrient diffusion and microbial mobility, resulting in limited degradation despite favorable thermal conditions. This study confirms that intramolecular-level isotopic techniques provide powerful tools for identifying microbial processes in low-permeability shales and contribute to a better understanding of shale oil and gas reservoir evolution.