(S)-Ornidazole is a subject of research as an antifertility agent in male animals at present. However, there seems to be no relative report on chiral separation for rac-Ornidazole, which has been used as an effective medicine for more than 30 years. In this article, the chiral separation of rac-Ornidazole on a Chiralcel OB-H column based on normal-phase high-performance liquid chromatography (NP-HPLC) is investigated and the methodology for detection of impurity of (R)-Ornidazole in (S)-Ornidazole injection and raw material is established. The novel mobile phase is utilized by mixing n-hexane, methanol and isopropyl alcohol (95:4:1, v/v/v) instead of the typical mobile phase of n-hexane and isopropyl alcohol, although the methanol, which offers a good resolution factor for the enantiomeric separation in this system, is not recommended on the Chiralcel OB-H column according to the instruction supplied by Daicel Chemical Ind., LTD (Japan).