BackgroundCircular　RNAs　(circRNAs)　have　emerged　as　a　prominent　class　of　covalently　closed　single-stranded　RNA　molecules　that　exhibit　tissue-specific　expression　and　potential　as　biomarkers　in　extracellular　vesicles　(EVs)　derived　from　liquid　biopsies.　Still,　their　characteristics　and　applications　in　EVs　remain　to　be　unveiled.MethodsWe　performed　a　comprehensive　analysis　of　EV-derived　circRNAs　(EV-circRNAs)　using　transcriptomics　data　obtained　from　1082　human　body　fluids,　including　plasma,　urine,　cerebrospinal　fluid　(CSF),　and　bile.　Our　validation　strategy　utilized　RT-qPCR　and　RNA　immunoprecipitation　assays,　complemented　by　computational　techniques　for　analyzing　EV-circRNA　features　and　RNA-binding　protein　interactions.ResultsWe　identified　136,327　EV-circRNAs　from　various　human　body　fluids.　Significantly,　a　considerable　amount　of　circRNAs　with　a　high　back-splicing　ratio　are　highly　enriched　in　EVs　compared　to　linear　RNAs.　Additionally,　we　discovered　brain-specific　circRNAs　enriched　in　plasma　EVs　and　cancer-associated　EV-circRNAs　linked　to　clinical　outcomes.　Moreover,　we　demonstrated　that　EV-circRNAs　have　the　potential　to　serve　as　biomarkers　for　evaluating　immunotherapy　efficacy　in　non-small　cell　lung　cancer　(NSCLC).　Importantly,　we　identified　the　involvement　of　RBPs,　particularly　YBX1,　in　the　sorting　mechanism　of　circRNAs　into　EVs.ConclusionsThis　study　unveils　the　extensive　repertoire　of　EV-circRNAs　across　human　biofluids,　offering　insights　into　their　potential　as　disease　biomarkers　and　their　mechanistic　roles　within　EVs.　The　identification　of　specific　circRNAs　and　the　elucidation　of　RBP-mediated　sorting　mechanisms　open　new　avenues　for　the　clinical　application　of　EV-circRNAs　in　disease　diagnostics　and　therapeutics.