A few cellular compartments maintain acidic environments in their interiors that are crucial for their proper function. Alteration in steady state organelle pH is closely linked to several diseases. Although a few probes exist to measure pH of cell compartments, each has several associated limitations. We present a high-performance pH sensor, a DNA nanoswitch, a convenient method to map spatiotemporal pH changes in endocytic pathways. DNA has been used to make a variety of nanoswitches in vitro . However, the present DNA nanoswitch functions as a pH sensing device equally efficiently intracellularly as it does in vitro . This DNA nanoswitch functions as a FRET-based pH sensor in the pH regime of 5.5–7, with high dynamic range between pH 5.8 and 7. It is efficiently engulfed by Drosophila hemocytes through endocytosis and can be used to measure the acidity of the endocytic vesicles that it marks during their maturation till their lysosomal stage.