Extracellular vesicles (EVs) including exosomes and microvesicles are nanometer-sized vesicles released by cells to deliver lipids, cellular proteins, mRNAs, and noncoding RNAs, thereby facilitating intercellular communication without direct cell-to-cell contacts. Due to their nanoscale size, EVs have been visualized under microscopy in vitro. We here describe a strategy to label EVs with Gaussia luciferase for noninvasive bioluminescence imaging and monitoring of systemically administered EVs in vivo.