RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping. The authors designed a chemical probe, azido-kethoxal, to specifically label guanosine in single-strand RNAs in live cells that could be used to determine transcriptome-wide RNA secondary structures.