Milk-derived extracellular vesicles (mEVs) are promising therapeutic delivery platforms due to their natural bioactivity, biocompatibility, and ability to cross biological barriers. However, analyzing their cellular uptake and trafficking is limited by existing fluorescent labeling methods, which often cause dye leakage and disrupt vesicle integrity. Here, a glycan-anchored fluorescence labeling strategy for mEVs is developed, involving periodate oxidation of surface sialic acids followed by aniline-catalyzed ligation of hydrazide-functionalized fluorophores. Nano-flow cytometry characterization confirmed ≈100% labeling efficiency without compromising mEVs integrity or uptake behavior. This approach enabled quantitative analysis of mEVs internalization, identifying clathrin-mediated endocytosis and macropinocytosis as the primary pathways and confirming mEVs’ capacity for lysosomal escape. Comparative analyses showed that traditional lipophilic dyes induced vesicle aggregation, dye leakage, and transfer, potentially misrepresenting mEVs behavior. Additionally, co-labeling mEVs with glycan-anchored fluorophores and FITC-conjugated paclitaxel enabled real-time tracking of drug delivery, revealing a burst release from lysosomes that led to significant cytotoxicity. Overall, the glycan-anchored fluorescence labeling allows precise analysis of mEVs uptake and intracellular fate, paving the way for further research and application in targeted drug delivery.