Exosome-based cell-free therapeutics has received increasing attention in recent decades. Due to the potential demand for therapeutic exosomes, appropriate methods for preservation and storage of exosomes are essential. Current cryopreservation strategies mostly focus on addition of cryoprotectants. However, due to the high concentration of cryoprotectant required, this approach can lead to unfavorable effects. Thus, other storage methods are urgently needed. In this study, we found that Tetraspanin 4 (TSPAN4) and other Tetraspanin family proteins play an essential role in protecting exosomes from cryo-damage. Moreover, we engineered TSPAN4-loaded exosomes which are resistant to cryo-damage. These engineered exosomes show similar properties to wild-type exosomes in protein composition, uptake by recipient cells, and cargo delivery efficiency. We believe our strategy of exosome cryopreservation, without the need for additional agents, will promote the clinical translation of exosomes as therapeutic agents.