Graphene, as a newly discovered carbon allotrope, has attracted broad interest and intense attention since its discovery for both fundamental research and a vast array of industrial and biomedical applications. Considerable efforts have been devoted to understanding the nano-bio-interfaces of graphene-based materials for exploring their potential biomedical applications, including drug delivery, biosensing, biomedical imaging, stem cell technology, and photothermal therapy. This review summarizes the current studies on the physiological stability, enhanced permeability and retention (EPR) effect, active targeting and drug carrying capability of graphene-based nanomaterials, and it provides a basic understanding about the mechanisms of drug and gene delivery by these nanomaterials. Also reviewed is the recent progress on photosensitizers and theranostics using graphene-based nanomaterials. The biosafety of graphene at the cellular and animal levels is discussed. The challenges and perspectives of the field are addressed.