Acidity in biological microenvironments plays a crucial role in physiology and pathology, so that pH monitoring can provide insights into the mechanisms underlying pH-related processes. Many pH ratiometric fluorescent probes have been developed based on intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) principles. In contrast, those based on intermolecular excimer are scarce. The present work rationally designed and readily synthesized a pyrene-derived ratiometric fluorescent probe (Pyrene-PA) for pH detection using the conversion of monomer-excimer emission. In an acidic aqueous medium, Pyrene-PA exists in a cationic monomer with an emission centered at 463 nm. As pH increases, Pyrene-PA converts to neutral molecules and exhibits reduced dispersibility, generating an intense excimer emission around 630 nm. Based on the above mechanism, Pyrene-PA displays a sensitive, selective, photostable, and reversible response to pH changes without interference from coexisting species such as anions, cations, thiols, and reactive oxygen species. The ratio of fluorescence intensity at 630 nm and 463 nm increases linearly with pH values from 4.0 to 7.0, and the pKa value is 5.6, suitable for pH quantification in acidic to neutral environments. Moreover, Pyrene-PA with low cytotoxicity and good cell penetrability enables monitoring pH changes in cells and zebrafish.