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A novel two-photon pH probe, 3-benzimidazole-7-hydroxycoumarin (BHC), was designed and synthesized based on the structures of hydroxycoumarin and benzimidazole. BHC showed good linearity in the pH ranges of 3.30–5.40 (pKa = 4.20) and 6.50–8.30 (pKa = 7.20) at a maximum emission wavelength of 480 nm. BHC in acidic and alkaline media could be distinguished by an obvious spectral shift of the maximum absorption wavelength from 390 nm to 420 nm. In addition, BHC was well localized to mitochondria and successfully applied to one-photon and two-photon imaging of pH changes in the mitochondria of HeLa cells. The findings presented herein suggest that BHC can serve as an excellent fluorescent probe for selectively sensing mitochondrial pH changes with remarkable photostability and low cytotoxicity. Unlabelled Image • A new fluorescent probe for one- and two-photon sensing of pH was developed. • The probe responded sensitively and selectively to pH changes within 3.0–6.0 (pKa = 4.20) and 6.0–9.0 (pKa = 7.20). • The probe was successfully applied for monitoring and imaging of mitochondrial pH in living cells. [ABSTRACT FROM AUTHOR]