A highly reactive ylide ^tBuA was introduced, which could react rapidly with the 5-formyl and 4-amino groups of 5-formylcytosine (5fC) under mild conditions without any co-solvent or catalyst in a manner of Wittig olefination and intramolecular nucleophilic substitution to yield a cyclized fluorescent adduct, which meets the demands of both single-base resolution sequencing and fluorescence switch-on detection of 5fC in DNA.