Potential of hydrogen (pH) is one of the most relevant parameters characterizing aqueous solutions. In biology, pH is intrinsically linked to cellular life since all metabolic pathways are implicated into ionic flows. In that way, determination of local pH offers a unique and major opportunity to increase our understanding of biological systems. Whereas the most common technique to obtain these data in analytical chemistry is to directly measure potential between two electrodes, in biological systems, this information has to be recovered in-situ without any physical interaction. Based on their non-invasive optical properties, fluorescent pH-sensitive probe are pertinent tools to develop. One of the most notorious pH-sensitive probes is fluorescein. In addition to excellent photophysical properties, this fluorophore presents a pH-sensitivity around neutral and physiologic domains. This review intends to shed new light on the recent use of fluorescein as pH-sensitive probes for biological applications, including targeted probes for specific imaging, flexible monitoring of bacterial growth, and biomedical applications.