There is a high demand to develop chemical tools to control the property and function of RNA. Current methods mainly rely on ultraviolet light-based caging strategies, which may cause phototoxicity in live cell-based experiments. We herein report an endogenous stimulus-responsive RNA acylation approach by introducing boronate ester (BE) groups to 2'-hydroxyls through postsynthetic modification. Treatment with hydrogen peroxide (H2O2) yields a phenol derivative which undergoes a 1,6-eliminaton for the traceless release of 2'-hydroxyl. We demonstrated that the acylation of crRNA enabled conditional regulation of CRISPR/Cas13a activity for activatable detection of target RNA. We also showed that the highly specific acylation of the single RNA in 8-17 DNAzyme allowed reversible control of the catalytic activity of DNAzyme, which was further applied to the cell-selective imaging of metal ions in cancer cells. Thus, our strategy provides a simple, general, and cell-selective method to control RNA activity, affording great potential in the construction of activatable RNA sensors and pre-RNA medicines.