Development of pH probes for intracellular pH detection is very important since changes in pH play an important role in physiological function. Fluorescein and its derivatives are one of the most widely used in modern biochemical, biological, medicinal, photochemical research due to their excellent photophysical properties. Fluorescein has different light absorption and fluorescence properties depending on pH since it has several acidic groups. Its protolytic equilibrium involves four species (ie, cation, neutral, monoanion, dianion) whose pKa values are approximately 2, 4, and 6.In this study, we synthesized new fluorescein derivatives from the reaction of 4-bromophthalic anhydride and resorcinol, and deoxyuridines labeled with a fluorescein derivative by Sonogashira coupling reaction. We will discuss their pH-dependent protolytic equilibria and photophysical properties in detail.