Probiotics are microorganisms beneficial to human health that can be engineered to perform various tasks, such as dietary supplementation, inflammation treatment, and even antitumor. Most probiotics need to overcome a hostile in vivo environment for successful colonization and survival; however, currently, no available strategies can be used to dynamically evaluate the survival or viability of probiotics in vivo, making their real use elusive. Herein, we develop a simple strategy to fabricate DNA nanodevice-modified lactobacilli for dynamically reporting probiotic viability, both in vitro and in vivo. To this end, a pH-responsive DNA nanodevice is designed, which is covalently immobilized on bacterial cells. Consequently, living lactobacilli continuously produce lactic acids and reduce the pH around cells, which activates the modified DNA nanodevices and generates strong fluorescence signals. Using this tool, we analyzed changes in the survival and viability of lactobacilli in a murine model, which holds great potential in the fields of food and medicine science.