Extracellular vesicles (EVs) are nanovesicles released by various cell types. EVs are known for cell-to-cell communications and have potent biological activities. Despite great progress in recent years for studies exploring the potentials of EVs for early disease detection, therapeutic application and drug delivery, determination of the favorable storage conditions of EVs has been challenging. The understanding of the impact of storage conditions on EVs before and after isolation is still limited. Storage may change the size, number, contents, functions, and behaviors of EVs. Here, we summarized current studies about the stability of EVs in different conditions, focusing on temperatures, durations, and freezing and thawing cycles. –80 °C seems to remain the most favorable condition for storage of biofluids and isolated EVs, while isolated EVs may be stored at 4 °C shortly. Lyophilization is promising for storage of EV products. Challenges remain in the understanding of storage-mediated change in EVs and in the development of advanced preservation techniques of EVs.