The sign and magnitude of permafrost carbon (C)-climate feedback are highly uncertain due to the limited understanding of the decomposability of thawing permafrost and relevant mechanistic controls over C release. Here, by combining aerobic incubation with biomarker analysis and a three-pool model, we reveal that C quality (represented by a higher amount of fast cycling C but a lower amount of recalcitrant C compounds) and normalized CO₂–C release in permafrost deposits were similar or even higher than those in the active layer, demonstrating a high vulnerability of C in Tibetan upland permafrost. We also illustrate that C quality exerts the most control over CO₂–C release from the active layer, whereas soil microbial abundance is more directly associated with CO₂–C release after permafrost thaw. Taken together, our findings highlight the importance of incorporating microbial properties into Earth System Models when predicting permafrost C dynamics under a changing environment.