Photodynamic therapy (PDT) is a promising noninvasive therapeutic technique and has attracted increasing interests in preclinical trials. However, the translation from laboratory to clinic often encounters the problem of undesired dark cytotoxicity of photosensitizers (PSs). Now, this challenge can be addressed by cascaded substitution activated phototheranostics using the host–guest strategy. Through electrostatical complexation of pyridinium-functionalized tetraphenylethylene, namely, TPE-PHO, and water-soluble calixarene, the dark cytotoxicity of TPE-PHO is dramatically inhibited. The nanoassemblies of the complex show enhanced biocompatibility and selectively locate at the cytoplasm in vitro. When TPE-PHO is competitively displaced from the cavity of calixarene by 4,4′-benzidine dihydrochloride at the tumor site, its dark cytotoxicity and photoactivity in tumor tissue are restored to give efficient PDT efficacy under light irradiation. The result from cell imaging reveals that TPE-PHO undergoes translocation from cytoplasm to mitochondria to kill the cancer cells during the cascaded supramolecular substitution process. In vivo tumor imaging and therapy are successfully implemented to evaluate the curative effect. Such a supramolecular strategy avoids tedious molecular synthesis and opens a new venue to readily tune the PS behaviors.