Real-time live cell imaging and quantification of biothiol dynamics are important for understanding pathophysiological processes. However, the design and synthesis of rational probes that have reversible and real-time capabilities is still challenging. In this work, we have prepared boron-dipyrrolemethene (BODIPY) based fluorescent molecules as ratiometric probes that allow the real-time biothiol dynamics to be observed in living cells. The Michael reaction between α-formyl-BODIPY (BOD-JQ) and GSH exhibited a reversible fluorogenic mechanism with fluorescent emission shifting from 592 nm to 544 nm with t1/2 = 16 ms. In particular, we showed that the probes with targeting agents are capable of detecting biothiols in mitochondria and the endoplasmic reticulum (ER) with high temporal resolution.