Cyproconazole, a chiral triazole fungicide, has been diffusely used and analyzed. The development of an effective analytical method for cyproconazole enantiomers can support their residual monitoring and risk assessment. In the present study, the absolute configuration of the cyproconazole enantiomers was confirmed by electronic circular dichroism and time-dependent density functional theory. The enantioseparation parameters were optimized by the response surface methodology using the Box-Behnken design on Lux Cellulose-2. The elution order of (2S,3R)-(+)-, (2S,3S)-(+)-, (2R,3S)-(-)-, and (2R,3R)-(-)-cyproconazole was simulated with molecular docking. The enantiomers were completely separated primarily via halogen bond and hydrogen bond interactions with the chiral stationary phases. The mean recoveries of the cyproconazole enantiomers in the four matrices were 71.8-102.0% with intraday relative standard deviations (RSDs) of 0.3-11.9% and interday RSDs of 0.9-10.6%. The results showed the chiral recognition mechanism clearly and confirmed that the method was accurate and convenient for the simultaneous detection of cyproconazole enantiomers in environmental and food matrices.