As one of three gasotransmitters, the fundamental signalling roles of hydrogen sulphide are receiving increasing attention. New tools for the accurate detection of hydrogen sulphide in cells and tissues are in demand to probe its biological functions. We report the p-nitrobenzyl-based ratiometric fluorescent probe RHP-2, which features a low detection limit, high selectivity and good photostability. The emission intensity ratios had a good linear relationship with the sulphide concentrations in PBS buffer and bovine serum. Our probe was applied to the ratiometric determination and imaging of endogenous H₂S in living cells. Furthermore, RHP-2 was used as an effective tool to measure endogenous H₂S in the mouse hippocampus. We observed a significant reduction in sulphide concentrations and downregulated expression of cystathionine β-synthetase (CBS) mRNA and CBS protein in the mouse hippocampus in a chronic unpredictable mild stress (CUMS)-induced depression model. These data suggested that decreased concentrations of endogenous H₂S may be involved in the pathogenesis of chronic stress depression.