Two-photon excited fluorescence microscopy and nanoparticle-assisted photodynamic therapy (PDT) are two important areas in biomedical research, and their combination can be more beneficial. A type of red emissive photosensitizer (PS) with aggregation-induced emission (AIE) features, which is called tetraphenylethylene (TPE-red), was synthesized and further encapsulated with poly(styrene-co-maleic anhydride) (PSMA) to form nanoparticles. Two-photon fluorescence, as well as two-photon excited reactive oxygen species (ROS) generation by TPE-red–PSMA nanoparticles, was characterized. A large two-photon absorption cross-section was observed at 1040 nm with a femtosecond (fs) laser. PDT via two-photon excitation was well realized on tumor cells, using TPE-red–PSMA nanoparticles as PSs under 1040 nm fs laser excitation. Based on our study, we believe that two-photon excited PDT with AIE-active PSs has great potential applications in deep tissue imaging-guided therapy.