The invention discloses a diclazuril enantiomer chiral chromatographic separation analysis method. The separation analysis method comprises the following steps of carrying out pretreatment on a chiralstationary phase, taking normal hexane, trifluoroacetic acid and lower alcohol as a mobile phase, and carrying out liquid phase chromatography separation analysis, wherein the pretreatment refers towashing of the chiral stationary phase by using the mobile phase, the washing time is not less than 2 hours, and the washing flow rate is not greater than 0.5mL.min-1; the chiral stationary phase is bonding type to parachloro-phenylcarbamoyl beta-cyclodextrin, coating type cellulose tri(3, 5-dimethyl phenylcarbamate) or coating type straight-chain starch tri(3, 5-dimethyl phenylcarbamate); the lower alcohol is ethanol, n-propyl alcohol, isopropanol or n-butyl alcohol; and the volume ratio of normal hexane to the lower alcohol is 50-80: 50-20. According to the chiral chromatographic separationanalysis method provided by the invention, the chiral stationary phase is pretreated, and the normal hexane, the trifluoroacetic acid and the lower alcohol are used as the mobile phase, so that the chiral chromatographic separation of the diclazuril enantiomer is realized.