Exosomes are bilayered vesicles derived from living cells and bacteria that are loaded with abundant biomolecules, such as proteins and nucleic acids. As an important medium of remote cell communication, exosomes are closely related to the occurrence and development of a number of diseases, including those involving tumors and inflammation. The isolation and enrichment of exosomes in complex biosystems is greatly significant for the diagnosis, prognosis, and detection of diseases, as well as in molecular-mechanism research. However, exosomes are usually nanoscale size distribution and widely existed in complex biological samples, including blood, tissue fluids, and urine, which bring difficulties and challenges to the isolation and enrichment of exosomes. To address this issue, several methods based on the physical properties of exosomes have been developed. For example, exosomes can be obtained by ultracentrifugation at high centrifugal force based on density differences; they can also be isolated and enriched by size-exclusion chromatography and ultrafiltration based on size heterogeneity. Exosomes can also be separated in high yields but with low purities using commercial polymer-coprecipitation-based isolation kits. While the abovementioned methods can be used to isolate and enrich exosomes in a highly efficient manner, accurately distinguishing interfering particles, including protein aggregates and microvesicles, in biosystems is still difficult, resulting in the poor purity of exosome isolation and enrichment. Affinity ligands are widely used during the affinity isolation and enrichment of exosomes. Antibodies exhibit high selectivity and affinity; consequently exosomes can be captured highly selectively by exploiting specific antigen/antibody interactions. However, antibodies also have some limitations, including complex preparation procedures, high costs, and poor stability. Chemical affinity ligands, such as aptamers, peptides, and small molecules, are also widely used to isolate and enrich exosomes. As a kind of molecular recognition tool, peptides contain a variety of amino acids and exhibit many advantages, including good biocompatibility, low immunogenicity, and design flexibility. Solid-phase synthesis strategies have rapidly developed, thereby providing a basis for automated and large-scale peptide synthesis. Affinity peptides have been widely used to recognize and analyze target biomolecules in complex physiological environments in a highly selective manner. A series of protein-targeting peptides has been reported based on the biomolecular characteristics of exosomes. These affinity peptides can be specifically anchored onto highly enriched transmembrane proteins on exosome surfaces, thereby enabling the efficient and highly selective isolation and enrichment of exosomes in complex systems. Additionally, exosomes contain stable bilayer membranes consisting of abundant and diverse phospholipid molecules. The development of phospholipid-molecule-targeting peptides is expected to effectively eliminate interference from protein aggregates and other particles. In addition to differences in the compositions of phospholipids in biofilms, exosomes are smaller and more curved than apoptotic bodies and microvesicles. A series of affinity peptides capable of inducing and sensing high membrane curvatures are widely used to isolate and enrich exosomes. The tumor microenvironment can produce and release tumor-derived exosomes that are buried in a large number of normal cell-derived exosomes. Accordingly, pH-responsive peptides have been designed and modified based on the acidic environments of tumor-derived exosomes, which were accurately and tightly anchored via peptide insertion and folding. Focusing on the current status of exosome research, this paper introduces and summarizes current and widely used methods for isolating and enriching exosomes. Various exosome-targeting peptide-design and screening principles are introduced based on the characteristics and advantages of peptides. The applications of these peptides to the isolation and enrichment of exosomes are also summarized, thereby providing strong guidance for the efficient and highly selective isolation and enrichment of exosomes in complex life-related systems.