Extracellular vesicles (EVs) are nanometer-sized particles naturally secreted by cells for intercellular communication that encapsulate bioactive cargo, such as proteins and RNA, with a lipid bilayer. Tumor cell-derived EVs (tdEVs) are particularly promising biomarkers for cancer research because their contents reflect the cell of origin. In most studies, tdEVs have been obtained from cancer cells cultured under static conditions, thus lacking the ability to recapitulate the microenvironment of cells in vivo. Recent developments in perfusable cell culture systems have allowed oxygen and a nutrient gradient to mimic the physiological and cellular microenvironment. However, as these systems are perfused by circulating the culture medium within the unified structure, independently harvesting cells and EVs at each time point for analysis presents a limitation. In this study, a modularized cell culture system is designed for the perfusion and real-time collection of EVs. The system consists of three detachable chambers, one each for fresh medium, cell culture, and EV collection. The fresh medium flows from the medium chamber to the culture chamber at a flow rate controlled by the hydraulic pressure injected with a syringe pump. When the culture medium containing EVs exceeds a certain volume within the chamber, it overflows into the collection chamber to harvest EVs. The compact and modularized chambers are highly interoperable with conventional cell culture modalities used in the laboratory, thus enabling various EV-based assays.