Ferroptosis is a pattern of cell death caused by iron-dependent accumulation of lipid peroxides and is closely associated with the occurrence and development of multiple diseases. Acrolein (ACR), one of the final metabolites of lipid peroxidation, is a reactive carbonyl species with strong biotoxicity. Effective detection of ACR is important for understanding its role in the progression of ferroptosis and studying the specific mechanisms of ferroptosis-mediated diseases. However, visualization detection of ACR during ferroptosis has not yet been reported. In this work, the first ratiometric fluorescent probe (HBT-SH) based on 2-(2'-hydroxyphenyl) benzothiazole (HBT) was designed for tracing endogenous ACR with an unprecedented regiospecific ACR-induced intramolecular cyclization strategy, which employs 2-aminoethanethiol as an ACR-selective recognition receptor. The experimental results showed that HBT-SH has excellent selectivity, high sensitivity (LOD = 0.26 μM) and good biocompatibility. More importantly, the upregulation of ACR levels was observed during ferroptosis in HeLa cells and zebrafish, indicating that ACR may be a specific active molecule that plays an essential biological role during ferroptosis or may serve as a potential marker of ferroptosis, which has great significance for studying the pathological process and treatment options of ferroptosis-related diseases.