At present, one of main problems for photo dynamic therapy (PDT) is how to improve the treatment depth. Two-photon activated (TPA) developed recently provide a possible solution for it. In this work, we report the energy-transferring assembled cationic dipeptide nanoparticles for two-photon activated photodynamic therapy (TPA-PDT). In the nanoparticles, the coencapsulated two-photon fluorescent dye bis(pyrene) (BP) is an energy donor, and a photosensitizer rose bengal (RB) is an acceptor based on an intraparticle fluorescence resonance energy transfer (FRET) mechanism. BP in the nanoparticles can be excited by one- or two- photon laser. And then, the energy of BP was transferred to RB, which highly enhanced the generation of singlet oxygen. The cellular experiments indicated that this nanosystem can induce the cytotoxicity under one- and two-photon irradiation, which PDT. allows further applications of FRET-based biomaterials for TPA-PDT.