The detection of small extracellular vesicles (sEVs) is currently a pivotal liquid biopsy approach for noninvasive cancer diagnosis. However, the lack of adequate specificity and sensitivity, as well as labor-intensive purification and analysis procedures, present challenges in isolating and profiling sEVs. Here, we present a protein-specific enzymatic optical reporter deposition-based liquid biopsy assay for the rapid and efficient capture and ultrasensitive detection of sEVs using a minimal volume of initial biofluids (10 μL). Biotin aptamers were employed to label sEV proteins for peroxidase conjugation, catalyzing the conversion of fluorescein tyramine into highly reactive free radicals. Efficient signal conversion was achieved by depositing nanoheterolayers composed of covalent tyraminated complexes onto sEV surfaces. The present method offers a detection limit of 6.4 × 10