BACKGROUND AND PURPOSE: Chemical 2, 4-dinitrofluorobenzene (DNFB), commonly called as Sanger’s reagent, is well known as skin sensitizer to cause dermatitis. However, how the DNFB causes skin inflammation remains unknown. In this study we aimed at identifying the molecular target that DNFB acts on. EXPERIMENTAL APPROACH: We used a fluorescent calcium imaging plate reader as an initial screening assay and patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was carried out to investigate DNFB binding sites in TRPA1 ion channel. KEY RESULTS: We found the chemical DNFB that selectively activates TRPA1 channel with EC50 of 2.36 ± 0.26 µM. Single-channel recording reveals that DNFB increases the channel open probability and acts on three residues C621, Y658 and E625 critical for DNFB-mediated TRPA1 activation. CONCLUSION AND IMPLICATIONS: Our findings not only explain a molecular mechanism underlying the dermatitis and pruritus caused by chemical DNFB, but also provides a molecular tool that is 7.5-time more potent than current AITC molecule and can be used for elucidating TRPA1 channel pharmacology and pathology.