Photodynamic therapy (PDT), employing photosensitizers to induce formation of reactive oxygen species (ROS) for tumor elimination, is emerging as a promising treatment modality in oncology due to its unique benefits. However, the PDT application in ovarian cancer, the most prevalent and lethal type of gynecological malignancy with a severe hypoxic microenvironment, remains unknown. This study revealed that photosensitizer TMPyP4 exhibited enhanced efficacy under H₂O₂ stimulation, with minimal change in cytotoxicity compared to TMPyP4 alone. The results showed that H₂O₂ increased ROS production induced by TMPyP4, leading to exacerbated mitochondrial dysfunction and DNA damage, ultimately inhibiting proliferation and inducing apoptosis in ovarian cancer cells. Mechanistically, H₂O₂ primarily enhanced the therapeutic efficacy of PDT with TMPyP4 against ovarian cancer cells by degrading HIF-1α, which subsequently modulated the HIF-1 signaling pathway, thereby alleviating the hypoxic environment in ovarian cancer cells. Our findings underscore the therapeutic potential of targeting HIF-1α within the hypoxic microenvironment for PDT in ovarian cancer and propose a novel integrated strategy for PDT treatment of this malignancy in vitro.