Hydrogen sulfide (H2S), a third gas signaling molecule, is considered to play a vital role in the development and treatment of diseases. To elucidate the intricate role of H2S in the organism and its participation in disease processes, there is an urgent need to visualize the dynamics of H2S. However, most currently available molecular probes have limitations in terms of sensitivity, specificity, and precision. In this study, the safe and biocompatible upconversion nanosensor NaYF4:Yb3+, Tm3+@NaYF4 anchored with Ag2O was successfully fabricated for H2S detection with an ultralow detection limit at 0.93 ng/mL. NaYF4:Yb3+, Tm3+@NaYF4@Ag2S is formed through in situ sulfuration reaction, and second near-infrared (NIR-II) fluorescence can be recorded upon the presence of H2S under 808 nm excitation. The results demonstrate the exceptional detection linearity and high specificity for H2S quantification. Additionally, NaYF4:Yb3+, Tm3+@NaYF4@Ag2O possesses a safe nature in normal and cancer cells. This nanosensor presents a NIR fluorescence imaging strategy for highly sensitive and specific detection of H2S, which has promises to be a practical tool for biomedical applications.