Conventional photosensitizers generally suffer from low efficiency in novel non-invasive two-photon photodynamic cancer therapy due to their small two-photon absorption cross section and they lack an imaging capability for therapy guiding due to their low fluorescence yield. Demonstrated here is the first water-soluble conjugated polymers as direct two-photon photosensitizers with dual capability of two-photon cell imaging and two-photon photodynamic therapy. By introducing a strong electron-withdrawing cyano group into the phenyl ring of the backbone, the cyano-substituted poly(fluorene-2,7-ylenevinylene-co-phenylene) (PFVCN) displays a 2.4 times higher maximum two-photon absorption cross section per repeat unit and significantly higher fluorescence quantum yield in water than the unsubstituted PFV. The large two-photon absorption cross section of PFVCN allows it to efficiently generate singlet oxygen under two-photon excitation, which is critical for two-photon photodynamic therapy. Two-photon excitation cell imaging and efficient two-photon-induced photodynamic therapy effect on cancer cells of PFVCN are successfully demonstrated. These studies provide insight in designing novel photosensitizing agents for simultaneous two-photon imaging and two-photon photodynamic therapy, which allows two-photon imaging guided therapy to fully take the unique advantages of two-photon excitation such as deep penetration and 3D selectivity.