Periodate (IO4-) is a widely used reagent in organic synthesis and for biomolecular modification. However, highly sensitive fluorescence detection methods for IO4- are lacking. In this work, we designed and synthesized iridium(III) complexes for the detection of IO4- by the incorporation of a methylthio group to serve as a luminescence trigger. The results showed that complex 1 could detect IO4- in a sensitive and selective manner with a detection limit of 0.077 mu M, rendering it more sensitive than reported fluorescence methods. Meanwhile, its inherently long emission lifetime enables its luminescence response to IO4- to be detected even in the presence of organic dyes by the use of time-resolved emission spectroscopy (TRES). This probe was further demonstrated to detect IO4- in living cells. To our knowledge, complex 1 is first long-lived iridium(III)-based probe for the sensitive detection of IO4-, and is also one of few existing imaging probes for IO4-.