Discovering the secrets of diseases from tear extracellular vesicles (EVs) is well-recognized and appreciated. However, a precise understanding of the interaction network between EV populations and their biogenesis from our body requires more in-depth and systematic analysis. Here, we report the biological profiles of different-size tear EV subsets from healthy individuals and the origins of EV proteins. We have identified about 1800 proteins and revealed the preferential differences in the biogenesis among distinct subsets. We observe that eye-related proteins that maintain retinal homeostasis and regulate inflammation are preferentially enriched in medium-size EVs (100 to 200 nm) fractions. Using universal analysis in combination with the Human Protein Atlas consensus dataset, we found the genesis of tear EV proteins with 37 tissues and 79 cell types. The proteins related to retinal neuronal cells, glial cells, and blood and immune cells are selectively enriched among EV subsets. Our studies in heterogeneous tear EVs provide building blocks for future transformative precision molecular diagnostics and therapeutics.