A novel fluorescent i-motif DNA silver nanoclusters system has been developed for visualization of reactive oxygen species in plants, enabling the detection of intracellular signaling in plant cells. Reactive oxygen species (ROS) are crucial in plant growth, defense, and stress responses, making them vital for improving crop resilience. Various ROS sensing methods for plants have been developed to detect ROS in vitro and in vivo. However, each method comes its own advantages and disadvantages, leading to an increasing demand for a simple and effective sensory system for ROS detection in plants. Here, we introduce novel DNA silver nanoclusters (DNA/AgNCs) sensors for visualizing ROS in plants. Two sensors, C20/AgNCs and FAM-C20/AgNCs-Cy5, detect intracellular ROS signaling in response to stimuli, such as abscisic acid, salicylic acid, ethylene, and bacterial peptide elicitor flg22. Notably, FAM-C20/AgNCs-Cy5 exceeds the sensing capabilities of HyPer7, a widely recognized ROS sensor. Taken together, we suggest that fluorescent i-motif DNA/AgNCs system is an effective tool for visualizing ROS signals in plant cells. This advancement is important to advancing our understanding of ROS-mediated processes in plant biology.