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Exosomes are nano-scale membrane-encapsulated vesicles produced by the majority of cells and have emerged as a rich source of biomarkers for a wide variety of diseases. Although many approaches have been developed for exosome isolation from biofluids, most of them have substantial shortcomings including long processing time, inefficiency, high cost, lack of specificity and/or surface marker-dependency. To address these issues, here we report a novel insulator-based dielectrophoretic (iDEP) device predicated on an array of borosilicate micropipettes to rapidly isolate exosomes from conditioned cell culture media and biofluids, such as plasma, serum, and saliva. The device is capable of exosome isolation from small sample volumes of 200 μL within 20 minutes under a relatively low (10 V cm -1 ) direct current (DC). This device is easy to fabricate thus, no cleanroom facility and expensive equipment are needed. Therefore, the iDEP device offers a rapid and cost-effective strategy for exosome isolation from biofluids in timely manner while maintaining the yield and purity.