Exosomes are cell-secreted nanoscale membrane vesicles that play critical roles in many pathophysiological processes. The clinical value of exosomes is under intense investigation, yet current knowledge regarding their in vivo properties is very limited because of the lack of efficient labeling techniques. Here, we report a phospholipid-based bioorthogonal labeling strategy to endow exosomes with optical probes without influencing their native biological functions. We investigated the dynamic in vivo biodistribution and organotropic uptake of multiple tumor exosomes in a single mouse. The results indicate that the exosomes derived from different cell lines show specific organotropic uptake. This phospholipid-based labeling strategy opens a new window to directly visualize and monitor exosome trafficking in living systems and holds great promise for exploring exosome-involved biological events such as cancer metastasis.