Photodynamic therapy (PDT) uses photosensitizers and visible light in combination with molecular oxygen to produce reactive oxygen species (ROS) that kill malignant cells by apoptosis and/or necrosis, shut down the tumor microvasculature and stimulate the host immune system. The excited singlet state of oxygen ( O ) is recognized to be the main cytotoxic ROS generated during PDT for the majority of photosensitizers used clinically and for many investigational new agents, so that maximizing its production within tumor cells and tissues can improve the therapeutic response, and several emerging and novel approaches for this are summarized. Quantitative techniques for O production measurement during photosensitization are also of immense importance of value for both preclinical research and future clinical practice. In this review, emerging strategies for enhanced photosensitized O generation are introduced, while recent advances in direct detection and imaging of O luminescence are summarized. In addition, the correlation between cumulative O luminescence and PDT efficiency will be highlighted. Meanwhile, the validation of O luminescence dosimetry for PDT application is also considered. This review concludes with a discussion on future demands of O luminescence detection for PDT dosimetry, with particular emphasis on clinical translation. Eye-catching color image for graphical abstract.