Exosomes are nanosized extracellular vesicles (EVs) that participate in intercellular communication through surface proteins and the delivery of internal cargo. The exosomes have gained attention for their potential as disease biomarkers and therapeutic agents. The therapeutic ability of exosomes has been verified by copious previous studies. Effective methods for extensive clinical applications are being researched for exosome-based regenerative therapies, including the application of 3D cultures to enhance exosome production and secretion which can resolve limited exosome secretory from the parent cells. Cell culture has emerged as a crucial approach for biomedical research because of its many benefits. Both well-established continuous cell lines and primary cell cultures continue to be invaluable for basic research and clinical application. Previous studies have shown that three-dimensional cultured exosomes (3D-Exo) improve therapeutic properties and yields compared to traditional culture systems. Since the majority of studies have focused on exosomes derived from mesenchymal stem cells (MSC-Exo), this review will also concentrate on MSC-Exo. In this review, we will summarize the advantages of 3D-Exo and introduce the 3D culture system and methods of exosome isolation, providing scientific strategies for the diagnosis, treatment, and prognosis of a wide variety of diseases.