Intracellular pH (pH_i) is a fundamental indicator of cellular physiological state, regulating cellular state and function, and has important research values. Although various probes for measuring intracellular pH were available, it is challenging to reflect pH_i in real-time and reversible manners. Herein, we developed a whole-cell bioluminescent (BL) probe based on wild type BL bacteria, photobacterium phosphoreum (P. phosphoreum), to determine and image pH_i. The dependence of BL intensity of P. phosphoreum on pH values of culture solutions was established. It was found that BL intensity could respond to the change of pH values rapidly and reversibly. We further revealed that P. phosphoreum maintained pH homeostasis in the extracellular pH (pH_e) within the range of 5.0–7.0, while intracellular pH homeostasis was destroyed at the alkaline pH_e. This method opens up the enormous potential of BL bacteria as an alternative to fluorescence for monitoring and imaging pH_i.