Exosome protein markers have garnered significant attention as liquid biopsy targets in recent years. They play a crucial role in early cancer diagnosis, treatment monitoring, and prognosis assessment. However, existing studies predominantly focus on all extracellular vesicles or all exosomes, failing to accurately distinguish and analyze tumor-derived exosome subpopulations directly associated with cancer. Consequently, the current accuracy and specificity of exosome proteomics research remain inadequate, severely limiting the efficiency of clinical protein marker screening. The primary challenges in exosome protein marker research include highly specific identification and separation of tumor-derived exosome subpopulations from complex serum samples while excluding interference from protein complexes and free proteins. To address these challenges, we employed the tumor marker epithelial cell adhesion molecule (EpCAM) as the recognition target for tumor-derived exosomes. We designed a corresponding nucleic acid aptamer and modified it onto the surface of liposomes. By fusing these liposomes with exosomes, we achieved precise identification and separation of tumor-derived exosome subpopulations within the serum background. Subsequent downstream proteomic analysis further enhances our understanding. This innovative approach enables relative quantitative analysis of serum proteomes from both prostate cancer patients and healthy individuals. It holds promise for accurate prostate cancer diagnosis and serves as a powerful tool for disease monitoring and prognosis assessment.