Understanding the interaction processes between the mainstream and its tributaries and detailing the rates of contribution of water and heat from two different water bodies in the tributary bay are essential for water management in the Three Gorges Reservoir (TGR). The stable isotope ratios of hydrogen (δD) and oxygen (δ18O) were applied to explore the interactions between the TGR mainstream and the tributary, Zhuyi River. A heat budget of the TGR tributary bay was constructed for the year 2014 using data for water temperature, meteorological data and water mixing ratio. The results showed that approximately 91% of the water in the tributary bay was from the TGR mainstream. And the heat budget reveals that the tributary bay net heat flux is dominated by the mainstream advective heat, and the average contribution rate is 87% during the whole year, while the tributary advective heat and atmosphere make a minor contribution to the tributary bay heat budget.