A highly selective imprinted sorbent was prepared by a surface molecular imprinting technique, and used for solid phase extraction-high performance liquid chromatography (SPE-HPLC) to determine trace norfloxacin (NOF) in complicated samples. The molecularly imprinted polymer (MIP) was obtained by using NOF as the template, β-cyclodextrin-methyl methacrylate (β-CD-MMA) and acrylamide (AM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as the cross-linking agent and toluene as the porogen. Various parameters affecting the extraction efficiency of the polymer have been evaluated to optimize the selective preconcentration of the NOF from fish samples. The NOF from fish samples, selectively extracted by MIP, was detected by RP-HPLC. The MISPE-HPLC method showed high selectivity and good recoveries (>85.7%). It was demonstrated that this MISPE-HPLC method could be applied for direct preconcentration and determination of quinolones in real samples.