Syphilis, caused by Treponema pallidum (T. pallidum), is associated with the oxidative stress due to its inflammation-like symptom, and detecting the reactive oxygen species (ROS) is crucial for monitoring the infectious process. Herein, we design and synthesize a perylene-based tunable fluorescent probe, PerqdOH, which can detect endogenous O2˙- during T. pallidum infection. The fluorescence peak shifted from 540 nm to 750 nm with increasing O2˙- levels. Besides, both decreased green fluorescence and enhanced red fluorescence could be observed simultaneously during the in vitro infection, providing the real-time monitoring of intracellular O2˙- caused by T. pallidum. Furthermore, the probe exhibited a remarkable signal in the treponemal lesions on the back of a rabbit model. Taken together, our synthesized PerqdOH holds great potential for application in clarifying the infectious process caused by T. pallidum in real time.