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In order to explore the re-aeration law of water flow over spillway, the transfer process of oxygen in water flow over spillway was studied. The interfacial mass transfer coefficients were obtained by experiments. The flow fields and the turbulence characteristics are simulated by numerical methods. The fractional volume of fluid model (VOF) of the air-water two phase flows was introduced to track the interface. Consequently, the quantitative expression of the interfacial mass transfer coefficients related with velocity and kinetic energy at the free surface was derived and the re-aeration model for the water flow over spillway was established. The examination with the experimental data of different conditions shows the validity of the re-aeration model for the water flow over spillways. This study will be important to evaluate the dissolved oxygen concentration and self-purification ability of rivers.