Based on the systematic review of the relevant laws, regulations and theoretical researches on the definition of stakeholders in the mechanism of ecological compensation of water flows, the framework of the rights and obligations of stakeholders is analyzed, and its actual compensation is described through the cases in water right transaction, water environment protection and flood storage and detention areas. The results show that: ①The China’s laws and regulations have clear definitions of the water flow ownership implemented by the State Council and the use rights obtained by the organizations and individual users, and their separation has been achieved through the water extraction permit system. ②The owner of water flows has the rights of their possession, use, profit and allocation based on the China’s laws and regulations. Furthermore, as the public interest spokesman of the natural resources, it is more responsible for the protection of the water flows. The users of water flows also have the obligations to protect them, and they should abide by the laws and regulations and ensure their quantity, quality and healthy eco-system. ③The mechanism of ecological compensation of water flows should be mainly aimed at the losses caused by encroaching the rights and interests of resources and environment ( development rights ) of the stakeholders who have the rights to use, and the contribution on the positive externalities of the ecological environment. ④It is necessary to further strengthen the government’s roles in the mechanism of ecological compensation of water flows, especially in allocating the compensation funds to the stakeholders who has the use rights of environmental resources and directly contribute to the water protection so as to establish the long-term mechanism. It is suggested that the ecological compensation in flood storage and detention areas should be carried out.