Thiol-containing amino acids (RSH) play a key role in maintaining the redox environment. In this work, we developed a novel dual-site two-photon (TP) fluorescent RSH probe (CA-TPP) for imaging mitochondrial RSH in living cells and mouse liver tissues. This probe exhibited rapid response and high TP photostability towards mitochondrial RSH. In particular, probe CA-TPP was capable of using TP fluorescence to track mitochondrial RSH over a long period.