The tetrazole-based photoclick chemistry has attracted considerable attention in virtue of its good biocompatibility, exclusive molecular reaction, and spatiotemporally controllable properties. Using this photoclick reaction, we designed an in situ, real-time fluorescence imaging system that targeted mitochondria and lysosomes in a spatiotemporally controllable manner. Upon irradiation, the pyrazoline fluorophore was generated in situ by the intramolecular tetrazole-alkene cycloaddition reaction (“photo-click chemistry”). This strategy exhibits features such as fast response, high efficiency, strong fluorescence intensity without background and superior stability. In addition, by integrating with an organelle-specific group, it has a good application for subcellular targeting imaging. Furthermore, the photo-responsive moiety Tet facilitates the probes, Mt-Tet and Ly-Tet, for the super-resolution imaging of subcellular structures.