Positioning essential elements of photodynamic therapy (PDT) to mitochondria can conquer the rigorously spatiotemporal limitation of reactive oxygen species (ROS) transfer and make considerable differences in PDT. However, precise accumulation of photosensitizer (PS) and oxygen within mitochondria is still challenging. Here, Hexyl 5-aminolevulinate hydrochloride (HAL) and 3-bromopyruvic acid (3BP) are simultaneously encapsulated into microparticles collected from X-ray irradiated tumor cells (X-MP). After systemic administration, the developed HAL/3BP@X-MP can specifically target and recognize tumor cells, where HAL induces the efficient accumulation of PpIX in mitochondria via the intrinsic haem biosynthetic pathway; meanwhile, 3BP remarkably increases the oxygen supply by inhibiting mitochondrial respiration. The accurate co-localization and prompt encounter of PpIX and oxygen produce sufficient ROS to directly disrupt mitochondria, resulting in significantly improved PDT outcomes.