Studies of organometallic reactions in living cells commonly rely on ensemble‐averaged measurements, which can obscure the detection of reaction dynamics or location‐specific behavior. This information is necessary to guide the design of bioorthogonal catalysts with improved biocompatibility, activity, and selectivity. By leveraging the high spatial and temporal resolution of single‐molecule fluorescence microscopy, we have successfully captured single‐molecule events promoted by Ru complexes inside live A549 human lung cells. By observing individual allylcarbamate cleavage reactions in real‐time, our results revealed that they occur with greater frequency inside the mitochondria than in the non‐mitochondria regions. The estimated turnover frequency of the Ru complexes was at least 3‐fold higher in the former than the latter. These results suggest that organelle specificity is a critical factor to consider in intracellular catalyst design, such as in developing metallodrugs for therapeutic applications.