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A magnetic molecularly imprinted polymer (M-MIP) for cyclobarbital (CY) has been prepared by a multi-step swelling and polymerization method using a uniformly-sized magnetic particle as the shape template. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high and low affinity sites, were formed on the M-MIP. The retention and molecular-recognition properties of the M-MIP toward CY, phenobarbital (PB), amobarbital (AM) and phenytoin (PT) were evaluated using a mixture of phosphoric acid and/or sodium phosphate buffer and acetonitrile as a mobile phase by LC. On the M-MIP, retention factors of CY, PB, AM and PT were drastically decreased at mobile phase pH 8.7. This could be ascribable to dissociation of these compounds, whose pKa values are around 8. The imprinting factors were in the order of CY > PB > AM > PT at mobile phase pHs 2.5-6.9, and those are very similar between CY and PB. Furthermore, they were decreased at mobile phase pH 8.7. In addition to shape recognition, hydrogen-bonding and hydrophobic interactions seem to work for the retention and molecular-recognition of CY, PB, AM and PT on the MIP. The M-MIP for CY was applied for the selective extraction of PB in human serum samples.