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In this reported study, a novel high-performance thin-layer chromatography (HPTLC) method was developed for the detection and quantification of the toxic substance di(2-ethylhexyl) adipate (DEHA) in Antarctic krill. This procedure was based on the extraction of DEHA by ultrasonic solvent extraction with anhydrous ethanol, silica-gel column chromatographic separation, HPTLC detection and quantification using petroleum ether/ethyl acetate/ acetone/glacial acetic acid (29:1:0.5:2d*, v/v/v/v) as the developing solvent and bromine thymol blue solution as the chromogenic agent. The content of DEHA in freeze-dried Antarctic krill was found to be ca. 0.63 ± 0.05 mg/g. The structure of DEHA in the Antarctic krill was subsequently determined by gas chromatography–mass spectrometry (GC-MS) and infrared chromatography, which verified the presence of this compound in the krill. The HPTLC method exhibited excellent accuracy, with a recovery of 97.1–101.6% and good precision with a relative standard deviation of 2.47–4.90%. The DEHA in Antarctic krill oil was extracted by n-hexane and detected using the same method described above, which verified that DEHA was also present in krill oil at a concentration of ca. 2.16 ± 0.08 mg/g. The presence of DEHA in kill oil is very concerning because of its demonstrated harmful ecotoxicity, and since Antarctic krill is the key link in the food chain in the Antarctic coastal marine ecosystem. The adverse effects of DEHA on Antarctic krill and the source of DEHA will be explored in future research.