We designed and prepared the imidazoline‐2‐thione containing OCl− probes, PIS and NIS, which operate through specific reactions with OCl− that yield corresponding fluorescent imidazolium ions. Importantly, we demonstrated that PIS can be employed to image OCl− generation in macrophages in a co‐culture system. We have also employed two‐photon microscopy and PIS to image OCl− in live cells and tissues, indicating that this probe could have wide biological applications. Imaging in a co‐culture system: The first two‐photon fluorescence probes for hypochlorite were developed. The imidazoline‐2‐thione probes operate through specific reactions with OCl− that produce fluorescent products. Using the probes, imaging OCl− generation in macrophages in a co‐culture system was demonstrated.