In this work, we studied the electrochemical activity of the graphene sheets embedded carbon (GSEC) film by doping oxygen. The GSEC film was prepared by electron cyclotron resonance (ECR) plasma sputtering system under electron irradiation with different electron acceleration voltage and oxygen content. Transmission electron microscopy and Raman confirm that oxygen atoms and electron irradiation induced more graphene edges and oxygen-containing functional groups on the surface. The electrochemical activities of the films were measured in the Fe(CN)64−/3− redox system and the results showed that a proper doping content of oxygen and electron acceleration energy is beneficial to enhance the electrochemical activity. The surface O-doped GSEC film with 5% content realizes the simultaneous detection of uric acid, xanthine and hypoxanthine. Furthermore, under the influence of electron acceleration energy, the film with 5% content showed high sensitivity in the simultaneous detection of sunset yellow and tartrazine. The mechanism could be that the isolated electron at graphene edge and the polar fraction of oxygen-containing functional groups provide more active sites to accelerate the electron transfer. These results indicated that surface O-doped GSEC films are promising electrode materials to construct sensitive electrochemical biosensors.