Abstract: The chiral separation of the racemic hexaconazole was obtained on amylose (phenylcarbamate) chiral column, which was prepared in authors' lab. The influence of chromatographic conditions on chiral separation was investigated. The results showed that the structure and concentration of alcohol in mobile phase could have a pronounced effect on enantiomeric separation and retention. In the range 15～40℃, a plot of lnα vs. 1/T of racemic hexaconazole was not a straight line. Between 15～30℃, the chiral separation was driven by enthopy. Between 30～40℃, the chiral separation was driven by enthalpy.