Disclosed are nucleic acid-based sensors for measuring the pH of a sample, including cells, regions thereof, and whole organisms. The sensor includes an I-switch that is triggered by protons, and which functions as a FRET-based pH sensor inside living cells and organisms. Also disclosed are compositions and methods for measuring the pH of a specific region of a cell, such as vesicles, the nucleus, mitochondrial matrix, or the Golgi lumen.