Extracellular vesicles are membrane-bound organelles that play crucial roles in intercellular communication and elicit responses in the recipient cell, such as defense responses against pathogens. In this study, we have optimized a protocol for isolating extracellular vesicles (EVs) from Sorghum bicolor apoplastic wash. We characterized the EVs using fluorescence microscopy and correlative light and electron microscopy. Key features • Allows the isolation of extracellular vesicles from the monocot plant Sorghum bicolor. • Labels isolated extracellular vesicles with fluorescent dyes for easy characterization with light microscopy. • Validates dye labeling and further characterizes extracellular vesicles using a correlative light and electron microscopy approach.