CD9 is a well-established sEV biomarker. However, the existing methods for isolating and detecting cancer-associated sEVs have several significant limitations that hinder their widespread adoption in research laboratories and clinical settings. Aptamers are single-stranded DNA/RNA molecules that adopt a specific three-dimensional structure, enabling them to bind with high affinity and specificity to target molecules. Through a peptide-based SELEX approach, we isolated DNA aptamers against a specific epitope on one of extracellular domains of human CD9. With several rounds of molecular engineering, we have evolved our CD9 aptamer to a ligand that binds to native CD9 proteins on the plasma membrane of both human cells and sEVs with high specificity. Utilising this newly developed CD9 aptamer, we developed a novel CD9 aptamer-based fluorescence polarization assay for the detection of cancer biomarker-positive sEVs that is also tetraspanin-positive. Impressively, our aptamer-based fluorescence polarization assay is capable of discriminating between sEVs originating from origins of cancers based on the quantitative difference of the biomarker molecules displayed on sEVs. Further refinement of our novel aptamer-guided liquid biopsy will pave the way for future personalized precision oncology.