Sediment respiration, the relation between dissolved oxygen (DO) attribution and carbon dioxide (CO2) emission, is an important index of the aquatic ecosystem and a key concern in the evaluation of reservoir cleanliness performance. To understand the sediment respiration dynamics regulated by the thermal stratification that is common in deep-water reservoirs, this study conducted in-situ measurements of thermal structures and benthic environments in the Daheiting Reservoir for 16 months. Then, the variations of DO and CO2 fluxes at the sediment-water interface (SWI) were obtained based on the aquatic eddy correlation method and the newly proposed virtual incubator method, respectively. The results show that the SWI fluxes dynamics can be decomposed into tendency variations dominated by thermal stratification and impulse variations induced by extreme events. The annual average SWI DO and CO2 fluxes of 3.80–6.62 and 1.92–3.15 mmol m−2·d−1 are estimated in the study site through the respiration dynamics, respectively, which CO2 flux is at a moderate level among the nearly 50 lakes and reservoirs worldwide but accounts for less than 15% of the total CO2 emission of this reservoir. Moreover, the sediment respiration quotient in stratified reservoirs is lower than in other aquatic environments, suggesting that the permanently flooded area is a weak net carbon source, while most carbon emissions from stratified reservoirs are carbon displacement or net carbon generated within the water. Sediment net carbon emissions correspond to human benefits such as flood control, power generation, and fisheries, whereas water net carbon emissions are usually not beneficial. Therefore, reducing net carbon emissions generated in the water may become an important way to achieve low-carbon operation of deep-water reservoirs.