Microvesicles (MVs) are an increasingly important source for biomarker discovery and clinical diagnostics. The small size of MVs and their presence in complex biological environment, however, pose technical challenges in sample preparation, particularly when sample volumes are small. We herein present an acoustic nanofilter system that size-specifically separates MVs in a continuous and contact-free manner. The separation uses ultrasound standing waves to exert differential acoustic force on MVs according to their size and density. By optimizing the design of the ultrasound transducers and underlying electronics, we were able to achieve a high separation yield and resolution. The "filter size-cutoff" can be controlled electronically in situ, which enables versatile MV-size selection. We applied the acoustic nanofilter to isolate nanoscale (<200 nm) vesicles from cell culture media as well as MVs in stored red blood cell products. With the capacity for rapid and contact-free MV isolation, the developed system could become a versatile preparatory tool for MV analyses.