Two-photon activated photodynamic therapy (TPA-PDT) is a recently developed technique that shows a potential for medical application. In contrast to traditional one-photon activated PDT, TPA-PDT can increase the treatment depth and decrease the damage to healthy tissue by using a near-infrared two-photon laser. However, this technique also suffers from the fact that approved photosensitive drugs have a low two-photon absorption cross section. In this study, it is demonstrate that doped polyglycerol mesoporous silica nanoparticles can carry a photosensitizer, Rose bengal, and can be applied in one- and two-photon PDT. TPA dye-doped mesoporous silica nanoparticles have been synthesized using a surfactant-free route, which can be considered a TPA-PDT platform after loading normal photosensitive drugs. The doped TPA dyes in the silica nanoparticles can transfer energy to the loading drugs via an intraparticle fluorescence resonance energy transfer (FRET) mechanism. The fluorescence lifetime and confocal laser scanning microscopy (CLSM) images obtained under different conditions demonstrated a FRET effect through both one- and two-photon activated modes. The results of cytotoxicity experiments proved that this TPA-PDT system could induce cellular apoptosis under one- or two-photon irradiation. This system in principle extends the application range of TPA-PDT.