Fluorescent imaging of cysteine (Cys) and homocysteine (Hcy) is crucial to understand sulphur metabolism in living cells. Whereas two-photon active cyclometalated Ir(III) complex for detecting Cys/Hcy has been rarely reported. Herein, two novel cyclometalated iridium (III) complexes Ir1-1r2 with different electron-donor were designed, synthesized and fully characterized as well as confirmed by single-crystal X-ray diffraction analysis, which could selectively react with Cys/Hcy, accompanying with an obvious one- and two-photon fluorescence enhancement in vitro. The mechanism for sensing Cys/Hcy was further analyzed in detail by mass spectra and time-dependent density functional theory calculations. It was found that Ir2, bearing ester group, exhibited higher performance on the selective detection of Cys and Hcy compared to In. Notably, the two-photon action cross-section value in the near-infrared region (similar to 800 nm) of Ir2 is significantly enhanced upon the addition of Cys/Hcy. Further application to cellular imaging indicated that Ir2 is membrane permeable and capable of monitoring the changes of intracellular mitochondrial Cys/Hcy. These results support that such two-photon active complexes might provide a promising platform for the detection of mitochondrial Cys/Hcy in living biological systems. (C) 2017 Published by Elsevier B.V.