Abstract: Tracking virus infection pathway in cells is of great significance for understanding the viral infection mechanism, and the successful labeling on viruses is the key to achieve viruses tracking. In this study, we proposed the in situ bioorthogonal strategy based on the bioorthogonal chemistry and metabolic engineering for overcoming defects of traditional virus labeling methods. Azido motifs were incorporated into virions through cell metabolism with lipid/amino acid/monosaccharide azide derivatives, and the modified virions were in situ labeled with dibenzocyclooctyne-functionalized fluorescent probes through the bioorthogonal reaction. The results show that viruses incorporated with azide motifs could be quickly and effectively captured by fluorescent probes to achieve viruses in situ labeling. Significantly, the in situ bioorthogonal labeling strategy is a general method for enveloped and non-enveloped viruses labeling with simple operations.