Vicinal dithiol-containing proteins (VDPs) are of considerable importance due to their role in regulating cellular functions through the reversible dithiol/disulfide inter-conversion reaction. However, there are currently no effective tools for the simultaneous tracing of endogenous VDPs in live cells. Here, we report an “off-on” fluorescent probe (RhQ) for the selective detection of VDPs and illustrate its utility for the long-term tracing of protein vicinal dithiols with simultaneous fluorescence imaging in various cell lines. Importantly, our study provides the first-time valuable insight into the localization of VDPs in whole cells, dynamically visualizing that presence of cell-surface protein vicinal dithiols in MCF-7 cells. Interestingly, some functional cell-surface VDPs have been reported for MCF-7 cells using proteomic analysis. Furthermore, the gradually released fluorescence from RhQ after its interaction with VDPs provides an overall picture of the cellular protein vicinal dithiol profile for different cell lines. This technology shows great promise as a visualization tool for revealing the role of cellular protein vicinal dithiols, especially VDPs on the cell-surface, and stimulating the design of VDP related drug candidates and vectors.