Graphene and its derivatives are increasingly applied in nanoelectronics, biosensing, drug delivery, and biomedical applications. However, the information about its cytotoxicity remains limited. Herein, the distribution and cytotoxicity of graphene oxide (GO) and TiO2-graphene oxide composite (TiO2-GO composite) were evaluated in A549 cells. Cell viability and cell ultrastructure were measured. Our results indicated that GO could enter A549 cells and located in the cytoplasm and nucleus without causing any cell damage. TiO2 nanoparticles and GO would be separated after TiO2-GO composite entered A549 cells. TiO2-GO composite could induce cytotoxicity similar to TiO2 nanoparticles, which was probably attributed to oxidative stress. These results should be considered in the development of biological applications of GO and TiO2-GO composite.