Abstract Fluorescent analysis of bone provides valuable insights into bone structures. However, conventional dyes suffer from low specificity on bone tissue, small stokes shift, short fluorescent lifetime, and aggregation‐caused quenching effect, which result in low efficacy and artifacts. In this work, we design an aggregation‐induced emission (AIE)‐active iridium(III) complex (Ir‐BP2) as a highly selective, convenient, nondestructiveness, and dual‐mode staining agent for bone analysis. Ir‐BP2 containing phosphonate groups selectively binds to hydroxyapatites, the main component of bone matrix, and exhibits turn‐on AIE phosphorescence with prolonged lifetime. Ir‐BP2 exhibits promising biosafety and offers higher accuracy in staining calcium deposits than conventional Alizarin Red S staining assay when it is employed in real‐time monitoring of osteogenesis differentiation process. A ready‐to‐use staining spray of Ir‐BP2 is fabricated. By using fluorescent imaging and lifetime imaging, Ir‐BP2 staining provides valuable insights into bone microstructure analysis, microdamage diagnosis, and bone growth state identification. Further, Ir‐BP2 is successfully applied on a human spine vertebra for diagnosing bone invasiveness of eosinophilic granuloma, validating its clinical practice. This work presents a powerful tool in bone analysis and will lead to new approaches for the diagnosis and treatment of bone‐related diseases.
