In order to clarify the sources and their seasonal variations of organic matter in surface sediment in the Three Gorges Reservoir region, the mass fractions of total organic carbon (TOC) and total nitrogen (TN) and their δ¹³C_org and δ¹⁵N isotopes in surface sediment samples from the mainstream and tributaries of the Three Gorges Reservoir region were measured in the summer and winter of 2017. The source ranges of organic matter were determined according to δ¹³C_org and the C/N ratio, with δ¹⁵N used for auxiliary analysis, and the Bayesian mixed model was used to quantify the contributions of different sources of organic matter in the Three Gorges Reservoir region. The results showed that the mass fractions of TOC and TN in surface sediment of the Three Gorges Reservoir region were 0.79% to 1.46% and 0.07% to 0.13%, respectively. The mass fraction of TOC in summer is slightly lower than that in winter, and that of TN has no seasonal difference. The ranges of δ¹³C_org and δ¹⁵N were -26.35‰ to -24.70‰ and 2.59‰ to 5.67‰, respectively, without seasonal difference. The organic matter of surface sediment in the Three Gorges Reservoir region mainly comes from soil organic matter and river plankton in summer, and soil organic matter and aquatic vascular plants dominate in winter. The proportion of sewage organic matter takes the fourth place in summer and winter, and further analysis with δ¹⁵N reveals that there is obvious external pollution in the Three Gorges Reservoir region, which is mainly related to artificial nitrogen fertilizer and domestic sewage, without obvious seasonal variation.