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Hydrogen peroxide (H₂O₂) is an important signaling molecule and plays key roles in multiple plant physiological processes. The rapid and direct monitoring of H₂O₂ could improve our understanding of its regulatory mechanisms in plants. In this study, we developed a paper-based analytical device consisting of a disposable nano-gold modified indium tin oxide working electrode to provide a platform for the rapid and direct detection of H₂O₂. The total analytical time was dramatically shortened to be approximate 3 min due to the avoidance of the time-consuming and complex treatment of plant samples. In addition, the amount of plant samples required was less than 3 mg in our approach. We used this system to monitor the concentrations of H₂O₂ in tomato leaves infected by <i>Botrytis</i><i>cinerea</i> within 24 h. Our results showed that the concentration of H₂O₂ in tomato leaves was increased in the initial phase, peaked at 1.5 μmol gFW⁻¹ at 6 h, and then decreased. The production trend of H₂O₂ in tomato leaves inoculated with <i>Botrytis</i><i>cinerea</i> detected with our approach is similar to the 3,3-diaminobenzidine staining method. Taken together, our study offers a rapid and direct approach for the detection of H₂O₂, which will not only pave the way for the further investigation of the regulation mechanisms of H₂O₂ in plants, but also promote the development of precision agriculture technology.