A high performance liquid chromatography method was developed to detect enantiomers of cypermethrin in pork muscle. Eight isomers of racemic cypermethrin were separated using a normal mobile phase of hexane–isopropyl alcohol (99.3–0.7, v/v) on a chiral CD-ph column in 25 min. The elution order of isomers was ascertained using enantiomer/diastereomer enriched cypermethrin standards. The extracts from pork samples were cleaned up by immunoaffinity column before detection. The recoveries from samples fortified levels of 0.1 and 1 mg kg−1 ranged from 67.3% to 113.5% with a relative standard deviation 6.7–16.8%; sufficient to effectively measure cypermethrin enantiomer concentrations in pig muscle tissue samples in the future.