Malignant　tumors　pose　a　serious　threat　to　human　life.　Dual-functional　agents　with　near-infrared　fluorescence　imaging　and　photodynamic　therapy　(PDT)　activities　have　significant　potential　for　synchronous　cancer　diagnosis　and　treatment　due　to　their　high　sensitivity　and　noninvasiveness.　In　this　study,　based　on　the　＇organelle-targeted　PDT＇　strategy,　we　developed　a　mitochondria-targeted　and　endoplasmic　reticulum　(ER)-located　fluorescent　probe,　HCy-BA,　which　was　constructed　by　the　introduction　of　the　phenylboronic　acid　group　in　the　receptor　component　and　the　α-β　unsaturated　ester　in　the　donor　component　of　hemicyanine.　HCy-BA　enables　near-infrared　fluorescence　imaging　of　ONOO-　and　viscosity　with　high　sensitivity　and　specificity,　and　can　effectively　distinguish　cancer　cells　from　normal　cells.　Meanwhile,　HCy-BA　exhibits　excellent　1O2　generation　capacity,　showing　significant　phototoxicity　toward　tumor　cells　and　effective　tumor　suppression　in　tumor-bearing　mice.　Furthermore,　HCy-BA　allows　real-time　monitoring　of　mitochondrial　autophagy　during　PDT,　facilitating　visualization　of　the　treatment　process　and　enabling　real-time　assessment　of　PDT.　These　results　highlight　the　potential　of　HCy-BA　in　cancer　diagnosis　and　treatment,　offering　new　insights　into　the　design　of　integrated　molecular　for　diagnosis　and　therapy　in　the　future.　©　2025　American　Chemical　Society.