Inflammation caused by traumatic, ischemic, infectious, autoimmune or toxic injury may further trigger cancer and even death. Overexpression of hydrogen sulfide (H2S) in vivo has been identified as a biomarker for various types of inflammation. Identification-responsive fluorescence imaging probes have broad application prospects for in vivo diagnosis of inflammation. However, it is a challenge to design an imaging probe that concurrently responds to the target molecules to improve the sensitivity and specificity of inflammation detection. Herein, we designed and synthesized an activatable two-photon fluorescent probe to detect H2S. Fl-H2S had high selectivity, excellent photostable signals and low detection limit for recognizing H2S. In addition, Fl-H2S showed excellent two-photon fluorescence properties in cell and liver tissue visualization experiments, with a penetration depth of up to 126 μm in liver tissue. Most importantly, the unique probe Fl-H2S was the first probe to monitor H2S levels in a mouse air pouch inflammation model by fluorescence imaging technology. We expect Fl-H2S to become an effective tool for longitudinal monitoring of inflammation, diagnosis of inflammation and prediction of underlying pathogenesis of related diseases by detecting H2S.