Objective To explore the use of bio?orthogonal click chemistry indocyanine green (ICG) labeling of adipose tissue?derived mesenchymal stem cells (ADSC) and in vivo cell tracking. Methods ADSCs were isolated and cultured, and then incubated with N?azidoacetylmannosamine?tetraacylated ( Ac4ManNAz) and Dibenzocyclooctyne?indocyanine green (DBCO?ICG) using bio?orthogonal click chemistry, and their cell viability was evaluated. ICG labeling was confirmed by laser confocal microscopy imaging. After ICG labeling, in vivo ADSC tracking was performed by near?infrared Ⅱ fluorescence imaging in acute liver injured mice. Liver tissue sections were also collected to analyze ADSC homing. Moreover, the therapeutic effects of ICG?labeled ADSCs on serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) and on pathological changes were also evaluated. Results ICG labeling of ADSCs could be achieved by bio?orthogonal click chemistry. Notably, ADSC viability and their therapeutic effects on acute liver injury, including serum ALT and AST and hepatic morphology, were not affected by this method. Near?infrared Ⅱ fluorescence imaging revealed the hepatic accumulation and homing of transplanted ADSCs in vivo and ex vivo. Conclusions Bio?orthogonal click chemistry may provide a promising new strategy for ADSC labeling and in vivo cell tracking.