Exosomes are membrane bound extracellular vesicles that play an important role in many biological processes. While they have great application value, exosome isolation is still considered a major scientific challenge. In the present study, a novel separation strategy for exosomes is proposed based on the specific interaction between immobilized peptide ligands and phosphatidylserine moieties which are highly abundant on the surface of exosomes. With the new affinity method, intact model exosomes can be recovered with a high yield in a short processing time. The purity of exosome samples enriched from serum by the affinity method is far higher than that isolated by ultrafiltration, and similar to that obtained by density gradient centrifugation and ultracentrifugation. Moreover, the variety of contaminants co-isolated by the affinity method is relatively low due to its specific separation principle. Proteomics analysis of exosomes isolated by the affinity method from the serum of healthy, hepatocellular carcinoma patients, and intrahepatic cholangiocarcinoma patients was performed to prove the applicability of this method. In conclusion, our novel strategy shows characteristics of easy preparation, high specificity, and cost-effectiveness, and provides a promising approach for exosome isolation which should have wide applications.