A novel rhodamine B-based fluorescent probe (RML) for lysosomal pH was developed by integrating a 4-(2-aminoethyl)morpholine moiety, which is a lysosome-targetable group, into a rhodamine B fluorophore, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. The probe responded to acidic pH at low concentration in a short amount of time. In addition, RML showed good membrane permeability and brilliant selectivity among various amino acids and metal cations. RML exhibited an 80-fold increase in fluorescence intensity at 583 nm throughout the pH range of 7.40-4.00 with a pKa of 5.16, which indicates that RML is valuable for studying intracellular acidic organelles. Moreover, RML has been successfully applied in HeLa cells, and the results demonstrated that RML could selectively stain lysosomes in living HeLa cells. Note that RML could be used to detect the pH increase in lysosomes induced by bafilomycin A1 within HeLa cells.