Photosensitizer doped conjugated polymer nanoparticles have been prepared by incorporating polyoxyethylene nonylphenylether (CO-520) into the nanoparticles using a re-precipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP) and an energy donor to enhance the two-photon excitation properties of TPP. These CO-520 incorporated, TPP-doped PFEMO nanoparticles are stable and have low cytotoxicity in the dark. The TPP emission of the nanoparticles was found to be enhanced by about 20 times by PFEMO under two-photon excitation. The nanoparticles showed significantly enhanced two-photon excitation singlet oxygen generation efficiency and two-photon photodynamic therapy activity in cancer cells. These composite nanoparticles display features required for ideal photosensitizers, such as low cytotoxicity in the dark and efficient two-photon photodynamic activity under laser radiation. In addition, these novel nano-photosensitizers allow simultaneous in vivo monitoring by two-photon fluorescence imaging during two-photon photodynamic treatment. These photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.