A new stationary phase for selectively recognizing gatifloxacin in aqueous media based on molecularly imprinted microspheres (MIMs) has been prepared by water/oil reverse micro-emulsion polymerization. The MIMs were prepared using gatifloxacin as the template, N, N'-methylenebisacrylamide as cross-linker and acrylamide and acryloyl-β-CD (β-CD-A, synthesized by ester reaction of acrylic acid with β-CD) as combinatorial functional monomers. The surface morphology of MIMs was characterized by scanning electron microscopy. The properties of MIMs recognition for gatifloxacin in water were studied by adsorption kinetics, adsorption isotherms combined with Scatchard analysis and selective recognition experiments. The results showed that the synthesized MIMs had an excellent ability to selectively recognize gatifloxacin in aqueous media. MIMs were employed as the chromatographic stationary phase to successfully separate the template gatifloxacin from its analogues. Discovering the mechanism of the MIMs recognition revealed that the memory cavities in the surface of the MIMs and hydrophobic effects between the template and the cavities of the β-CD residues were the primary contributions to the special recognition process.