The flexible and freestanding graphene oxide (GO) film was fabricated for drug delivery and antibacterial. The film was synthesized by covalently attaching cefalexin onto graphene oxide sheets and then made by filtration of the colloidal suspension. SEM and optical images show that the Cefalexin-grafted graphene oxide (GO-CE) film possesses the unique 2D layer-by-layer structure and it could form channels for drug release when immersed in water. The drug loading and release tests certify that the GO-CE film is a promising drug delivery membrane with high load capacity (0.621 mg mg -1 ) and long-acting release properties (72 h), and can effectively inhibit the growth of E. coli and S. aureus bacteria while showing minimal cytotoxicity for a long time. The cellular culture results of the HeLa Cells indicate that the GO-CE film exhibits excellent biocompatibility. Based on these advantages, the GO-CE film is expected to be used in the environmental and medical applications.