Photodynamic therapy is a noninvasive medical procedure in which malignant tissue is damaged by reactive oxygen species generated from targeted light irradiation on a photosensitizer. Herein, we incorporate an Ru(II)-based two-photon photosensitizer into a macrocyclic structure via coordination-driven self-assembly. Properties favorable for photodynamic therapy arise upon formation of the metallacycle, including near-infrared luminescence, strong two-photon absorption, high reactive oxygen species generation efficiency, and selective accumulation in mitochondria and nucleus that are hypersensitive to reactive oxygen species, resulting in good photodynamic therapy efficacy in both in vitro and in vivo investigations. This study shows that supramolecular coordination complexes provide a platform for efficient tuning on the photophysical and biological properties of molecules with biomedical interest.