Dissolved organic carbon (DOC) plays an important role in permafrost carbon cycle. However, substantial uncertainties remain about what determines the biodegradability of permafrost‐derived DOC and how the biodegradability varies between permafrost and active‐layer soils at a regional scale. Here, we conducted an incubation experiment with DOC leached from permafrost and active‐layer soils along a ~1,000‐km permafrost transect on the Tibetan Plateau. Our results showed that permafrost‐derived DOC had high biodegradability, with an average of 21.7% loss of DOC over a 28‐day incubation. The biodegradability of DOC leached from permafrost soils was larger than or equal to that in active‐layer within 72% of sampling sites. Our results also revealed that variation in DOC biodegradability in permafrost soils and its difference with that in active‐layer soils were jointly determined by vegetation types, soil properties, and related DOC composition. The biodegradability of permafrost‐derived DOC in alpine steppe and meadow was lower than that in swamp meadow. It also declined with the decreasing soil carbon: nitrogen ratio, increasing pH, clay content, DOC molecular weight, and its degradation degree. Similarly, the variability in the difference of DOC biodegradability between permafrost and active‐layer soils also resulted from the differences of three types of above‐mentioned factors between the two layers. These results suggest that permafrost thawing‐induced DOC release could intensify permafrost carbon‐climate feedback due to its vulnerability to microbial decomposition, with its impact depending on vegetation, soil, and substrate properties.