Enzymes containing 3'-5' exonuclease activities play vital roles in maintaining genome stability. Though a wide variety of methods have been developed for detection of these enzymes, few of them can be directly applied for in situ and real-time monitoring of the secretion of these active substances by living cells. Taking advantages of the free 3'-end of stacked guanine-quenched photoinduced electron transfer fluorescent probes, here we demonstrate a novel assay capable of in situ and real-time monitoring of the 3'-5' exonucleases secreted by living cells. The detection limit of the new method achieved as low as 0.04 U/mL, allowing direct monitoring of the target enzymes in an extracellular environment without preconcentration steps. False positive signals caused by other nonspecific enzymes were easily ruled out by the use of a control probe with the 3'-end modified with exonuclease-resistant phosphorothioate guanines. Using Alexa Fluor 488 as the fluorophore, the probe is adaptable to a wide range of pH conditions. The approach was successfully applied for in situ, real-time monitoring of the 3'-5' exonucleases secreted by suspension cells of Arabidopsis thaliana. It also holds great potential for in situ and real-time detection of many other DNA end-processing enzymes produced by other types of cells.