objective: Phosphatidylserine (PS)-deficient small extracellular vesicle (sEV) subpopulations (PS sEVs) circulate in blood for long periods; hence, they are expected to have therapeutic applications. However, limited production of PS sEVs makes their application difficult. In this study, a method for the preparation of such populations using an enzymatic reaction was developed. methods: Bulk sEVs collected from a cell culture supernatant via ultracentrifugation were subjected to an enzymatic reaction using phosphatidylserine decarboxylase (PSD). The yield of PS sEVs was estimated using magnetic beads that bind to PS sEVs. Then, the physical properties and pharmacokinetics (PK) of the sEVs were evaluated. results: Enzymatic depletion of PS exposed on sEV surfaces using PSD increased the yield of PS sEVs. PSD treatment hardly changed the physicochemical properties of PS sEVs. Moreover, the serum concentration profile and PK parameters of the PS sEVs derived from PSD-treated bulk sEVs indicated a long blood-circulation half-life. conclusions: Treatment of sEVs with PSD successfully reduced surface PS levels and increased the amount of the PS sEV subpopulation among bulk sEVs. This protocol of efficient preparation of PS sEVs based on PSD treatment, as well as information on the basic PK, can be foundational for the therapeutic application of sEVs.