H2S donors in living cells are essential for modulating H2S levels and have been proposed to be relevant for managing hepatic disorders, but conventional platforms to screen for H2S donors are plagued by interference by endogenous background fluorescence signals. Here, the authors develop a luminogenic probe—based on an Ir(III) complex with a 1,10-phenanthroline-5,6-dione moiety—capable of selective response to mitochondrial H2S, and set up an anti-interference high-throughput screening system capable of distinguishing target signals from complex background autofluorescence in living cells.