Environment‐responsive in situ synthesis of molecular fluorescent dyes is challenging. Herein, we develop a photoextension strategy to make trimethine cyanines with decent conversion efficiency (up to 81 %) using 1‐butyl 2,3,3‐trimethyl 3H‐indole derivatives as the sole precursors, and demonstrate a free radical mechanism. In the inducer‐extension stage, free radicals and reactive oxygen species (ROS) were able to mediate similar reactions with no assistance of light. We explored a Mito‐extension strategy to in situ synthesize trimethine cyanines in the living cells. The cellular ROS‐dependence provided a foundation for preferential cyanine expression in cancer cells. Finally, we applied an iodized precursor as an intrinsic ROS‐activated theranostic agent that integrated mitochondria‐targeted cyanine synthesis, cell imaging and phototherapy. [ABSTRACT FROM AUTHOR]