Small extracellular vesicles (SEVs) secreted from various cells are lipid bilayer vesicles, 30-150 nm in size, that carry proteins, nucleic acids, and lipids as cargos to other cells. They include exosomes, which are generated in multivesicular endosomes (MVEs) and secreted upon fusion of MVEs with plasma membranes and a part of microvesicles, which directly bud from plasma membranes. SEVs have attracted attention as diagnostic and drug discovery targets, since it has been demonstrated that SEVs are involved in the intercellular communication in many diseases and physiological phenomena such as cancer, neurodegenerative diseases, and immunity. There are five isolation methods for SEVs, which include ultracentrifugation, density gradient ultracentrifugation, polymer precipitation, affinity isolation, and size-exclusion chromatography. The affinity isolation, which isolates SEVs using magnetic beads conjugated with binding molecules such as antibodies, has the ability to isolate highly pure SEVs in character. However, the population of SEVs is limited by the binding molecules and it is difficult to elute intact SEVs from the antibody beads. In this chapter, we present a TIM4-affinity isolation method that targets phosphatidylserine (PS), a component of the SEV membrane. TIM4 binds to PS in a Ca2+-dependent manner, which enables the elution of intact SEVs from TIM4-beads in the presence of the chelating reagent ethylenediaminetetraacetic acid (EDTA). The TIM4-affinity isolation method helps overcome the disadvantages of the affinity isolation method and enables the isolation of heterogeneous SEVs at high purity. This method will facilitate the functional analysis of SEVs, development of diagnostic methods, and drug development of engineered SEVs.