Abstract: The characteristics of hydrogen and oxygen stable isotopes in river is important for regional hydrologic cycle research. To uncover water supply sources in subtropical agricultural basin from a perspective of stable isotopes, field measurements were conducted in four reaches (S₁, S₂, S₃ and S₄) of Tuojia River from April to August 2017. We analyzed the spatial and temporal variations in hydrogen and oxygen isotopes and deuterium excess parameters and their relationship with precipitation, altitude and water quality. Results showed that hydrogen and oxygen isotopes and deuterium excess values ranged from -43.17‰ to -26.43‰ (-35.50‰±5.44‰), -7.94‰ to -5.70‰ (-6.86‰±0.74‰), and 16.77‰ to 23.49‰ (19.39‰±1.95‰), respectively. Under the influence of monsoon circulation, hydrogen and oxygen isotopes showed substantial seasonal variation, with spring (δD: -29.88‰±3.31‰; δ¹⁸O: -6.18‰±0.57‰) > summer (δD: -39.25‰±2.65‰; δ¹⁸O: -7.32‰±0.42‰). The spatial distribution of hydrogen and oxygen isotopes values increased fluctuantly with the position from the sampling site to the river’s source, with δD: S₁<S₄<S₃<S₂, and δ¹⁸O: S₁<S₃<S₄<S₂. The deuterium excess values had no significant temporal variation, while it spatially increased gradually with the river levels. The slope and intercept of water line in this river were smaller than that of the local meteoric water line, suggesting that precipitation was the primary water source for this river. At the seasonal scale, both δD and δ¹⁸O were significantly negatively correlated with water temperature (δD: r=-0.92; δ¹⁸O: r=-0.88) and δ¹⁸O was negatively correlated with altitude (r=-0.96). At spatial scale, δ¹⁸O had a significantly positive correlation with water temperature. The δD and δ¹⁸O had negative correlation with precipitation, but being not statistically significant.