Benzoyl peroxide (BPO), as a widely used organic peroxide, has attracted widespread attention from all sectors of society for its environmental hazards and potential risks to human health. Herein, we employed a Förster resonance energy transfer (FRET) strategy to construct a novel ratiometric fluorescent probe CY-DCI for BPO detection in food, zebrafish, and mice. Specifically, a hemicyanine fluorophore and a dicyanoisophorone fluorophore were connected with a piperazine group as donor and acceptor, respectively, and an olefinic unsaturated bond as the reaction site. CY-DCI has favorable selectivity and an excellent detection limit as low as 58.1nM, and the recovery rates for real-sample detection ranged from 95.8% to 104%, with relative standard deviations (RSD) less than 2.58%. To further improve its practicality, silica gel plates and test strips containing CY-DCI (0-50μM) were developed for naked-eye detection of BPO with satisfactory results. Additionally, this novel probe was then applied for ratiometric imaging of living zebrafish and mice and showed high ratiometric imaging resolution in the green and red channels, thus demonstrating its practical application for BPO detection and toxicity early warning in food and biosystems. Environmental ImplicationFirstly, Benzoyl peroxide (BPO) affects soil microbial activity, thereby disrupting soil structure and function. Secondly, BPO leads to the death of aquatic organisms and destroys the balance of the aquatic ecosystem. Thirdly, BPO may cause damage to human tissues and induce related diseases. In addition, BPO is highly flammable and may cause explosive decomposition. In short, BPO is harmful to the environment and should be used with care and better managed. Therefore, it is important to develop a new and efficient method and technology for the rapid detection of BPO.