A novel mitochondrion-specific live-cell fluorescence turn-on photoactivatable bioprobe, named o-TPP3M, is designed and readily prepared, operating in a new photoactivatable mechanism based on a twisted intramolecular charge-transfer effect, the restriction of intramolecular rotations, and photocyclodehydrogenation. This bioprobe exhibits unique photoactivation behavior, a large signal-to-background ratio, and no cytotoxicity to living cells, providing a promising candidate for varied bioresearch.