Photosensitive fluorescent probes have become powerful tools in chemical biology and molecular biophysics, which are used to investigate cellular processes with high temporal and spatial resolution. Accordingly, photosensitive fluorescent probes, including photoactivatable, photoconvertible, and photoswitchable fluorophores, have been extensively developed during the past decade. The photoswitchable fluorophores have received much attention because they highlight cellular events clearly. This minireview summarizes recent advances of using reversibly photoswitchable fluorophores and their applications in innovative bioimaging. Photoswitchable fluorophores include photoswitchable fluorescent proteins, photoswitchable fluorescent organic molecules (dyes), and photoswitchable fluorescent nanoparticles. Several strategies have been developed to synthesize photoswitchable fluorophores, including engineering combination proteins, chemical synthesis, polymerization, and self-assembly. Here we concentrate on polymer nanoparticles with optically switchable emission properties: either fluorescence on/off or dualalternating- color fluorescence photoswitching. The essential mechanisms of fluorescence photoswitching enable different types of photoswitchable fluorophores to change emission intensity or wavelength (color) and thus validating the basis of the fluorescence on/off or dual-color photoswitching design. Generally the possible applications of any fluorophores are to label biological targets, followed by specific imaging. The newly developed photoswitchable fluorophores enable super-resolution fluorescence imaging because of their photosensitive emission. Finally, we summarize the important area regarding future research and development on photoswitchable fluorescent nanoparticles.