A selective phosphorescent biothiols probe is synthesized based on Ir(III) complex 1, which has 2,2′-biquinoline as the N^N ligand for realizing the satisfied two-photon absorption cross-section and two-functionalized 2-phenylpyridine ligands with an α,β-unsaturated ketone moiety as the thiol reaction site. The one- and two-photon optical properties of 1 are investigated through UV–vis absorption spectrum and photoluminescence spectrum. This Ir(III) complex can act as an excellent one- and two-photon excited “OFF–ON” phosphorescent probe for biothiols based on the 1,4-addition of biothiol to α,β-unsaturated ketones. Moreover, one- and two-photon-induced luminescent imagings of biothiols in living cells are also realized. Furthermore, the experiments of time-resolved photoluminescence technique and fluorescence lifetime imaging microscopy demonstrate that 1 is able to detect biothiols in the presence of strong background fluorescence. In addition, probe 1 is adsorbed into the shell of mesoporous silica nanoparticles with core–shell structure to form a nanoprobe, which can realize the ratiometric detection of biothiols in absolute water solution and living cells based on two phosphorescent signals.