Herein, highly selective surface molecularly imprinted polymers (SMIPs) were prepared on the surface of silica microspheres and employed as a new stationary phase for high performance liquid chromatography (HPLC). In this paper, myricetin was selected as the template and successfully grafted onto the derivatized silica particles (size: 5 μm), and acetonitrile/methanol were used as the porogen and co-solvent. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and thermo-gravimetric analysis (TGA) were used as characterization measures, and the results illustrated that the polymers were successfully synthesized. The obtained uniform polymer particles were packed in stainless steel columns (250 mm × 4.6 mm) and evaluated as the imprinted stationary phase. Then the packed columns were connected to an Agilent 1100 series HPLC system to separate a mixture of four flavonoids. The SMIP stationary phase showed a good effect on the separation of the flavonoid mixture.