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40 reviewsGlycosylated RNAs (glycoRNAs), a recently discovered class of membraneassociated glyco-molecules, remain poorly understood in function and clinicalvalue due to limited detection methods. Here, we show a dual recognitionFörster resonance energy transfer (drFRET) strategy using nucleic acid probesto detect N-acetylneuraminic acid-modified RNAs, enabling sensitive, selectiveprofiling of glycoRNAs on small extracellular vesicles (sEVs) from minimalbiofluids (10 μl initial biofluid). Using drFRET, we identify 5 prevalent sEVglycoRNAs derived from 7 cancer cell lines. In a 100-patient cohort (6 cancertypes and non-cancer controls), sEV glycoRNA profiles achieve 100% accuracy(95% confidence interval) in distinguishing cancers from non-cancer cases and89% accuracy in classifying specific cancer types. Furthermore, drFRET revealthat sEV glycoRNAs specifically interact with Siglec proteins and P-selectin,which is critical for sEV cellular internalization. The drFRET strategy provides aversatile and sensitive platform for the imaging and functional analysis of sEVglycoRNAs, with promising implications for clinical applications.