The multiphoton absorbing (MPA) materials have been extensively investigated due to their crucial merits of long wavelength excitation and short wavelength emission, of which the organometallic complexes are the most promising but still challenging candidates for developing efficient MPA materials. Herein, a novel cyclometallated iridium complex (abbreviated as SQ) is reported as a multiphoton absorption phosphorescent probe, which is constructed by coordinating the central metal Ir with diethoxyphenyl terpyridine, phenyl pyridine and SCN ligands. The single crystal of SQ exhibits a twisted octahedral structure that can avoid phosphorescence quenching caused by π-π stacking, and the push-pull electron effect of the ligand endows SQ with excellent nonlinear optical (NLO) properties under near-infrared excitation. As a result, two-photon imaging with deep tissue penetration depths is realized at the excitation wavelength of 750 nm. Meanwhile, the planar conjugated structure of diethoxyphenyl terpyridine facilitates the insertion of SQ into base pairs of mitochondrial DNA (mtDNA), thus limiting the intramolecular rotation. Consequently, the emission intensity of the SQ solution enhanced approximately 10-fold after mtDNA was introduced, which led to mtDNA-specific recognition. Our work proves a new way of designing a phosphorescence probe that can specifically identify mtDNA, thus realizing dynamic monitoring of mitochondria.