As conventional bulky methods for extracellular vesicle (EV) separation are unsuitable for small volumes of samples, microfluidic devices are thought to offer a solution for the integrated and automatic processing of EV separation. This study demonstrates a simple microfluidic aqueous two-phase system (ATPS) for EV separation with high recovery efficiency to overcome the limitation of previous devices, which require complex external equipment or high cost manufacturing. With polyethylene glycol and dextran in the microfluidic channel, the isolation mechanism of the microfluidic ATPS was analyzed by comparison between two-phase and one-phase systems. Our device could facilitate continuous EV isolation with 83.4% recovery efficiency and remove 65.4% of the proteins from the EV-protein mixture. EVs were also successfully isolated from human plasma at high recovery efficiency.