We have developed a highly promising new probe named Mito-pH2, which can be used for both colorimetric and fluorescent-based pH detection. This donor-acceptor dye validates the use of the phenolic group in the donor part of the dye for generating a sensitive pH probe in the pH range between 3 and 9. Spectroscopic analysis confirmed the mechanism for pH sensing. Mito-pH2 shows dual excitation and dual emission, thereby enabling ratiometric based response in-vitro. Mito-pH 2 emits in the red region and shows low cytotoxicity making it highly suitable for live cell imaging. Mito-pH2 specifically localises to the mitochondria in live cells. We also demonstrated its efficacy in sensing dynamic mitochondria pH changes in cells, wherein putative pH changes triggered by extracellular addition of either acid or base could be sensed by Mito-pH2. Interestingly, Mito-pH2 also revealed considerable heterogeneity in the measured mitochondria pH in live cells. Heterogeneity was seen both across cells and even within a single cell across the mitochondrial network. Overall, this work shows Mito-pH2 has significant potential as a valuable tool for real-time monitoring and understanding of mitochondrial pHrelated cell biology.