Extracellular vesicles (EVs) have emerged as promising delivery vehicles for RNA-based therapeutics because of their advantages over other mRNA delivery systems, including their excellent biosafety and biocompatibility, stability against degradation, and the ability to cross physiological barriers such as the blood-brain barrier. In this study, we report a nanosecond electroporation (nsEP) system with a microfluidic configuration. Applying millisecond and nanosecond pulses separately shifted the main impact of electroporation from the cell membrane to the membrane structure of cellular organelles. After being treated by a nsEP system, mouse embryonic fibroblasts (MEFs) demonstrated the capability to generate large quantities of small extracellular vesicles (sEVs). To gain a better understanding of the biological mechanisms underlying this nsEP-triggered sEV release, we conducted a proteomics analysis.