Hydrogen sulfide (H2S) is an important gaseous signaling agent mediated by many physiological processes and diseases. In order to explore its role in biological signaling, much effort has been focused on developing organic fluorescent probes to image H2S. However, these downconversion H2S probes are impractical for bio-imaging beyond a certain depth because of the short tissue penetration of UV/visible light (as an excitation source). In most circumstance, these probes are also not suitable for long-term assay due to photo-bleaching. Herein, a new design to detect H2S based on the coumarin-hemicyanine (CHC1)-modified upconversion nanophosphors is reported. This inorganic–organic integrated nanoprobe is demonstrated to display a fast response time with a large ratiometric upconversion luminescence (UCL) enhancement, and extraordinary photo-stability. CHC1-UCNPs not only can be used for ratiometric UCL monitoring of pseudo-enzymatic H2S production in living cells, but can also be used to identify the risk of endotoxic shock through ratiometric UCL imaging of tissue and measurement of endogenous H2S levels in plasma. The first ratiometric UCL H2S nanoprobe reported here may be further developed as the next-generation diagnostic tool for the detection of inflammatory-related diseases.