Photodynamic therapy is a highly promising method for cancer adjuvant treatment. However, the current research on the microscopic changes during the photodynamic therapy process is still quite limited, which seriously impedes the deep understanding of the procedure. For this purpose, a novel polarity-responsive probe, MI-PPF, with excellent mitochondrial targeting and anchoring capabilities has been rationally designed and synthesized. Notably, MI-PPF has successfully realized the in situ detection of mitochondrial morphology and polarity alterations during the photodynamic therapy process in cancer cells through fluorescence lifetime imaging. The results showed that a series of phenomena such as deformation, shrinkage, vacuolation, and aggregation occurred in the mitochondrial morphology during photodynamic therapy. Concurrently, a decline in mitochondrial polarity is also noted, which may be closely linked to the mitochondrial oxidative stress response during this process. Furthermore, MI-PPF can be used for photodynamic therapy on tumor mouse models and has successfully achieved fluorescence lifetime imaging of tumor sections before and after photodynamic therapy, uncovering multifaceted changes in cell morphology, polarity, and polarity distribution within the mouse tumor model during the process. It is anticipated that this study will offer valuable insights and guidance to the research of mitochondrial-related fields and will boost the advancement of diagnostic and therapeutic areas for associated diseases.