Biothiols are the main antioxidants in regulating the redox balance and resisting oxidative stress in various biological processes, but the long detection time of current fluorescent probes hinders their rapid imaging in vitro and in vivo. To reveal the influx of biothiols, we rationally develop an ortho -activation approach to accelerate the reaction between the probe and biothiols, by introducing electron-withdrawing fluorine atom into the ortho -site of the phenolic hydroxyl group in the NIR probe to generate an ortho -inductive effect. The ortho -fluorine helps to increase the chemical reactivity of the molecular structure, resulting in a significantly shorter detection time (within 5 min) as compared to previous reports (> 20 min for acrylates-based probes in aqueous solution). Based on this approach, our near-infrared probe 2F-RBX can sensitively and efficiently detect endogenous biothiols in living HepG2 cells and in vivo. These data suggest that ortho -activation is a simple and flexible approach to construct sensitive fluorescent probes for rapid imaging of biothiols, and perhaps other molecules in future, under biological circumstances. [Display omitted] • Rational design of an ortho -activation strategy to develop a NIR probe. • A novel turn-on NIR fluorescent probe rapidly detecting biothiols. • Capable of detecting endogenous biothiols in living cells. • Applicable to in vivo imaging in living mice. [ABSTRACT FROM AUTHOR]