Celiprolol enantiomer was resolved directly by using normal-phase HPLC with urea derivative as chiral stationary phase (CSP). The resolution condition was optimized by varying the content of ethanol and 1, 2-dichloroethane in the mobile phase. The effects of the two components on stereoselectivity factor (alpha) and stereochemical resolution factor (Rs) are demonstrated. The higher the 1,2-dichloroethane content the faster the elution of solute is, but the lower the values of alpha and Rs are. For a suitable content of ethanol in mobile phase, the maximum resolution factor (Rs) can be obtained. Ethanol is a strong proton-donor and proton-acceptor. Its strong hydrogen bond interaction with solute and CSP is important for the direct resolution. In order to obtain both the low retention time and a high Rs, we chose the mobile phase with n-hexane:1,2-dichloroethane:ethanol (V/V/V) = 77:21:2. Other organic modifiers such as methanol, iso-propanol, n-butanol and acetonitrile were also used. Iso-propanol, methanol and n-butanol showed longer retention time and lower values of alpha and Rs than ethanol. Acetonitrile is only a proton-acceptor and has weak hydrogen bond interaction with solute and CSP, so resolution wasn't obtained. The elution order of enantiomer was also discussed. We thought that the hydrogen bond interaction between solute and the (S)-val component of CSP may mainly control the chiral recognition. The interaction strength is different between (R)- and (S)-celiprolol, so the celiprolol enantiomer was resolved. And the elution order is S, R.