Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (similar to 1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.