Photosensitizers (PSs), featuring switchable properties to achieve tunable reactive oxygen species (ROS) generation, are highly desirable in photodynamic treatment and antibacterial applications. A supramolecular strategy combined with aggregation-induced emission (AIE) characteristics for synergistic design of an effective PS has emerged as a promising approach to reduce its side effects. Herein, we have constructed a ternary supramolecular PS based on a two-step Förster resonance energy transfer (FRET) process using an AIE-active pillar[5]arene host, a spiropyran derivative (SP-G) guest, and Nile blue (NiB) dye in aqueous media. The reversible isomerization process of SP-G endows this PS with the “on-off” switchability. Under UV light irradiation, the guest SP-G (non-emissive closed-form) can be converted to merocyanine MC-G (emissive open-form). And the in situ formed MC-G can function as the first energy acceptor and an efficient PS for ROS generation. Meanwhile, the introduction of NiB promotes a synergistic ROS-generation activity, leading to a two-step dynamic FRET process in response to UV and visible light irradiation. Moreover, this photoswitchable system with excellent controllable fluorescence performance and ROS-generation ability can be applied to inactivation of cancer cells and bacteria. The present work on the basis of a supramolecular strategy provides a proof-of-concept and new insight for the fabrication of smart PS materials.