A new ratiometric fluorescent probe based on Förster resonance energy transfer (FRET) for sensing lysosomal pH has been developed. The probe (RMPM) was composed of imidazo1,5-αpyridine quaternary ammonium salt fluorophore as the FRET donor and the rhodamine moiety as the FRET acceptor. It’s the first time to report that imidazo1,5-αpyridine quaternary ammonium salt acts as the FRET donor. The ratio of fluorescence intensity of the probe at two wavelengths (I 424 /I 581 ) changed significantly and responded linearly toward minor pH changes in the range of 5.4–6.6. It should be noted that it’s rare to report that a ratiometric pH probe could detect so weak acidic pH with pKa = 6.31. In addition, probe RMPM exhibited excellent water-solubility, fast-response, all-right selectivity and brilliant reversibility. Moreover, RMPM has been successfully applied to sensing lysosomal pH in HeLa cells and has low cytotoxicity.