Polytetrafluoroethylene (PTFE) has been widely applied as a biomaterial owing to its good stability and bioinertness. A PDA/TiO2 composite film was prepared on the surface of PTFE substrate through polydopamine (PDA) assisted in-situ co-deposition, so that the hydrophilicity and cellular responses could be improved. It was found that the film consists of PDA and TiO2. The content of TiO2 in the film can be controlled through the amount of TiO2 sol added in the deposition solution. The film is bonded to the substrate strongly. The shear strength reaches 23.5 MPa. Fibroblasts and osteoblasts are seeded on the surface of the modified substrate, and one-day cell adhesion and three-day proliferation experiments show that the cellular response of the modified substrate are significantly improved. Such deposition method is simple and easy, exhibiting wide potential in surface modification of PTFE for biomedical application.