Anti-seasonal variations in water levels in the Three Gorges (TG) Reservoir have led to significant changes in the reservoir's hydrological environment. To explore the response of water metabolic processes in the reservoir under different water level fluctuation scenarios, their responses to environmental changes, and the carbon sequestration and emission in the water body, this study selected two transects (Cuntan transect and Shaiwangba transect) located within the TG reservoir, which are affected by different impounding degrees, and one transect (Zhutuo transect), located outside the TG reservoir, which is unaffected by impounding processes, along the main stream for comparison research. The Cuntan transect is located in a fluctuating backwater area and is affected by high water-level scenarios from the TG reservoir. The Shaiwangba transect is affected by water storage from the TG reservoir year-round. The Zhutuo transect is a natural river transect. Based on high-frequency monitoring of hydrological and hydrochemical data collected in 2023, water metabolism was calculated using the single-station open channel method. A molecular diffusion model was employed to estimate carbon flux in the water body. Additionally, a random forest model was applied to rank the environmental factors influencing net ecosystem production (NEP) according to their importance. The results showed: ① Gross primary productivity (GPP) was the highest at Cuntan, followed by Zhutuo and Shaiwangba, while respiration (ER) was the highest at Shaiwangba, followed by Zhutuo and Cuntan. Due to reservoir impoundment, the Shaiwangba transect exhibited the lowest photosynthetic efficiency and the highest respiratory efficiency among the three transects. ② The environmental factors driving NEP changes at each transect were similar, with dissolved oxygen (DO), water temperature, partial pressure of carbon dioxide (pCO2), photosynthetically active radiation (PAR) and pH being the primary contributing factors. ③ There was no clear correlation between water level and NEP changes at the Shaiwangba transect. The Zhutuo transect showed an opposite trend between the water level and NEP. The Cuntan transect exhibited an inverse trend between water level and NEP during the flood season and the initial impoundment phase, while it was no clear pattern during the later stages of impoundment, high water levels, and drawdown periods. ④ The Cuntan transect was autotrophic system in the spring season, exhibiting a significant carbon sink effect, and was heterotrophic system in other seasons, showing a significant carbon source effect. Both Zhutuo and Shaiwangba transects were heterotrophic systems throughout all seasons, with a prominent carbon source effect. These findings contribute to understanding the mechanisms of the water environment and carbon cycle changes driven by water level fluctuations in large reservoirs and are of significant importance for water environment protection and carbon cycle process research in reservoirs.