Herein, a novel pH-responsive phosphorescent probe based on cyclometalated iridium(III) complex is reported. To prevent oxygen quenching of phosphorescence and to improve the probe biocompatibility, the complex is covalently conjugated with a water-soluble block-copolymer that also increases its pH sensitivity. The resulting polymeric nanoprobe demonstrates a strong response of the phosphorescence lifetime onto pH variations in physiological range. Cellular experiments with Chinese hamster ovary (CHO-K1) cells show the predominant internalization of the probe in acidified cell compartments, endosomes and lysosomes. The analysis of phosphorescence lifetime imaging microscopy data confirms applicability of the sensor for monitoring of intra- and extracellular pH in cell cultures.