<p>Extracellular vesicles (EVs) are lipid bilayer-enclosed nanovesicles that mediate intercellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids. Tissue-derived EVs (Ti-EVs) have gained increasing attention due to their ability to reflect the physiological or pathological state of the tissue microenvironment. Ti-EVs are generally classified into two types: tissue-sourced EVs (Ts-EVs) and tissue-explant-derived EVs (Te-EVs). Although both types originate from tissues, they differ in isolation methods, biological characteristics, and research applications. This review systematically compares the isolation procedures, methodological advantages and limitations, and research progress of Ts-EVs and Te-EVs. Furthermore, we summarize their applications across various organ systems, focusing on their protein and RNA cargo. Finally, we discuss current challenges and future directions in Ti-EVs research.</p>