<p>Exosomes are small extracellular vesicles originating from the inward budding of endosomal membrane discharged into the extracellular space during exocytosis. Exosomes are essential for facilitating communication between cells, presenting antigens, transferring proteins, mRNAs, and miRNAs. These functions have significant implications for both diagnostic and therapeutic applications. Hence, it is imperative to devise a straightforward, effective and economical technique for extracting exosomes to facilitate scientific research and medical diagnostics. Our study involved the comparison of two prominent methods for isolating exosomes, ultracentrifugation (UC) and precipitation by total exosome isolation (TEI) reagent, as indicated by the biophysical and physicochemical properties of serum-derived exosomes. The presence of exosomes was demonstrated by an array of techniques, including high-resolution scanning electron microscopy, high-resolution transmission electron microscopy, zeta potential and sizer and FTIR. We employed nanoparticle tracking analysis to evaluate the purity and quantity of the isolated exosomes. Our findings indicate that the TEI technique leads to greater exosomal yield, recovery, and purity, which can be directly translated into drug delivery and targeted therapeutics. Ultracentrifugation effectively conserved exosome morphology; however, it resulted in particle aggregation and reduced the average size of the produced exosomes. Furthermore, the expression of let-7a-5p determined using RT-qPCR was significantly greater in the exosomes derived from the TEI group than in those derived from the UC group. The objective of our comparison study is to assist researchers and clinicians in selecting the most suitable exosome isolation techniques for subsequent use.</p> Graphical Abstract <p></p>