Abstract: The water source of subsurface flow on a slope of alpine meadow in the Qinghai Lake watershed, China, was identified with the Deuterium and Oxygen-18 isotope values of precipitation, soil water and subsurface flow during April to September in 2013 based on a two-source model. Results showed that the contribution of precipitation to soil water was higher in surface soil than that in deeper soil. The isotope values of subsurface flow were closer to those of deeper soil water, which meant that subsurface flow was mostly derived from the deeper soil water. In addition, the average contribution ratios of soil water to subsurface flow on the upper slope and the middle slope in non-rain periods were 88.54% and 78.43% respectively. But during the rainfall events, the main water source of subsurface flow changed from soil water to rainfall and changed back to soil water after rainfall. It was concluded that soil water played a critical role in water source of subsurface flow.