Controlled intracellular chemical reactions to regulate cellular functions remain a challenge in biology mimetic systems. Herein, we developed an intra-mitochondrial bio-orthogonal reaction to induce aggregation induced emission. In situ carbonyl ligation inside mitochondria drives the molecules to form nano-aggregates with green fluorescence, which leads to depolarization of the mitochondrial membrane, generation of ROS, and subsequently mitochondrial dysfunction. This intra-mitochondrial carbonyl ligation shows great potential for anticancer treatment in various cancer cell lines.