The significance of H₂O₂ as a marker of reactive oxygen species (ROS) and oxidative stress in living organisms has spurred growing interest in its roles in inflammation and disease progression. In this report, a ratiometric time-gated luminescence (RTGL) probe is proposed based on mixed lanthanide complexes, ER-BATTA-Tb³⁺/Eu³⁺, for imaging the H₂O₂ generation both in vitro and in vivo. Upon exposure to H₂O₂, the probe undergoes cleavage of the benzyl boric acid group, releasing hydroxyl (─OH) groups, which significantly reduces the emission of the Eu³⁺ complex while slightly increasing the emission of the Tb³⁺ complex. This response allows the I₅₄₀/I₆₁₀ ratio to be used as an indicator for monitoring the H₂O₂ level changes. The probes are capable of selectively accumulating in the endoplasmic reticulum (ER), allowing effective imaging of H₂O₂ in the ER of living cells and liver-injured mice under oxidative stress. Moreover, by integrating ER-BATTA-Tb³⁺/Eu³⁺ into (polyethylene glycol) PEG hydrogels, the H₂O₂-responsive smart sensor films, PEG-H₂O₂-Sensor films, are created, which enable the real-time monitoring of H₂O₂ levels in various wounds using a smartphone imaging platform and R/G channel evaluation. The sensor films are also innovatively applied for the in situ monitoring of H₂O₂ in brains of epileptic rats, facilitating the precise assessment of brain damage. This study provides a valuable tool for the quantitative detection of H₂O₂ in vitro and in vivo, as well as for the clinical monitoring and treatment of H₂O₂-related diseases in multiple scenarios.