The tetramethylrhodamine (TMR) fluorophore is a useful platform for fluorescence probes, being applicable, for example, to biological investigations utilizing fluorescence microscopy, owing to its excellent photochemical properties in aqueous media. We have developed new TMR derivatives that show different dependences of their behavior upon the environment. Among them, HMTMR showed unique characteristics, and its putative spirocyclic structure was confirmed by X-ray crystallography. Utilizing this discovery, we have established a strategy to modulate the fluorescence of TMR by regulating the spirocyclization, and we have obtained a new fluorescence probe that can detect hypochlorous acid specifically. This probe, HySOx, can work in 99.9% aqueous solution at pH 7.4 and was confirmed to be able to detect hypochlorous acid being generated inside phagosomes in real time. HySOx is tolerant to autoxidation and photobleaching under bioimaging conditions. Regulation of the spirocyclization of rhodamines, as we describe here, provides a new approach to the rational development of novel fluorescence probes.