Dissolved organic carbon (DOC) plays an indispensable role in biogeochemical cycles and ecosystem services in rivers. However, little is known about the seasonal variations of DOC biodegradation in subtropical rivers. Here, we investigated the concentrations of DOC, dissolved total nitrogen (DTN), and dissolved total phosphorus (DTP), humification index (HIX), fluorescence index (FI), and DOC biodegradation in 57 rivers in the dry and wet seasons in the Three Gorges Reservoir area, China, and the aims were to clarify the temporal changes in DOC biodegradation and its driving factors in these subtropical rivers. Compared with dry season, DTN and DTP concentrations, and HIX value were greater, and FI value was lower in the wet season. However, DOC biodegradation remained unchanged across the two sampling seasons. Further, DOC biodegradation negatively correlated with DOC:DTP ratio, DTN:DTP ratio, and FI in the dry season, but only with HIX in the wet season. These findings emphasis that, despite unchanged DOC biodegradation, the key factors driving DOC biodegradation shift from C:N:P stoichiometry in the dry season to initial chemical composition in the wet season in subtropical rivers. Our results regarding the temporal patterns of DOC biodegradation and the underlying mechanisms bear important implications for a better understanding of C dynamics in subtropical river ecosystems.