This review provides a comprehensive overview of the current state of exosome-based strategies in nanomedicine and regenerative medicine. Small extracellular vesicles (sEVs), particularly exosomes, have gained significant attention due to their unique properties, such as stability, biocompatibility, and minimal immunogenicity. These properties make them ideal for various biomedical applications, including cancer therapy, immunotherapy, and biomarker research. However, the practical utilization of sEVs is hindered by expensive isolation procedures, limited circulation lifetime, and suboptimal targeting capacity. The review highlights the importance of understanding current isolation, loading, and characterization techniques to overcome these limitations. It also emphasizes the potential of exosomes in regenerative medicine, where they can modulate cellular functions and promote tissue regeneration. The biogenesis, isolation, and characterization of exosomes are discussed in detail, along with advanced isolation techniques like microfluidics and single-EV analysis methods. Exosomes have been explored for drug delivery in various disorders, including brain, lung, liver, and other cancers. They have shown promise in treating neurodegenerative disorders, respiratory illnesses, and liver diseases. Additionally, exosomes are being used in immunotherapy to enhance immune responses and improve cancer treatment outcomes. Despite the progress, challenges remain, such as the high cost of isolation techniques and the need for more efficient loading methods. The review concludes by emphasizing the potential of exosomes in expanding their applications in nanomedicine and regenerative medicine, with ongoing clinical trials highlighting their therapeutic potential.This review provides a comprehensive overview of the current state of exosome-based strategies in nanomedicine and regenerative medicine. Small extracellular vesicles (sEVs), particularly exosomes, have gained significant attention due to their unique properties, such as stability, biocompatibility, and minimal immunogenicity. These properties make them ideal for various biomedical applications, including cancer therapy, immunotherapy, and biomarker research. However, the practical utilization of sEVs is hindered by expensive isolation procedures, limited circulation lifetime, and suboptimal targeting capacity. The review highlights the importance of understanding current isolation, loading, and characterization techniques to overcome these limitations. It also emphasizes the potential of exosomes in regenerative medicine, where they can modulate cellular functions and promote tissue regeneration. The biogenesis, isolation, and characterization of exosomes are discussed in detail, along with advanced isolation techniques like microfluidics and single-EV analysis methods. Exosomes have been explored for drug delivery in various disorders, including brain, lung, liver, and other cancers. They have shown promise in treating neurodegenerative disorders, respiratory illnesses, and liver diseases. Additionally, exosomes are being used in immunotherapy to enhance immune responses and improve cancer treatment outcomes. Despite the progress, challenges remain, such as the high cost of isolation techniques and the need for more efficient loading methods. The review concludes by emphasizing the potential of exosomes in expanding their applications in nanomedicine and regenerative medicine, with ongoing clinical trials highlighting their therapeutic potential.