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Since its introduction in the 1990s, two\u2010photon excited microscopy has spurred the rapid emergence of novel 2PE\u2010based applications, including photodynamic therapy (PDT) and the targeted release of biologically active agents. This review first elucidates the fundamental principles of 2PE before comprehensively examining the evolution of photosensitizers (PSs) for two\u2010photon PDT (2P\u2010PDT), covering both nanomaterials and small molecules, categorized based on their respective characteristics. Additionally, it explores advancements in PS development for integrating 2P\u2010PDT with other therapeutic modalities (e.g., photothermal therapy, chemotherapy, immunotherapy, gene therapy, etc.). Subsequent sections provide an overview of photoactivatable compounds with two\u2010photon absorption properties for the controlled release of various therapeutic agents (e.g., drugs, genes, and gasotransmitters), emphasizing their potential in anticancer applications. 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