Spiramycin I is a main component of spiramycin and a structural analogue of neospiramycin I, which is an impurity in spiramycin production. Molecular imprinting technology (MIT) has many advantages in selectivity and environmental compatibility in biomolecular separation. In this study, core-shell molecularly imprinted polymers (MIPs) were prepared for spiramycin I. Methyl methacrylate (MMA) was used to prepare the seed core, and acrylamide (AM)/methacrylic acid (MAA) was chosen to prepare the shell. The structure of polymerized MIPs was successfully characterized by transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and elemental analysis. The adsorption results showed that MIPs polymerized by different monomers exhibited very different adsorption for spiramycin I. MIPs with monomeric AM achieved a fast mass transfer, high adsorption capacity and high selectivity. Then, these MIPs were applied to separate spiramycin I from the fermentation broth in the chromatographic column, and its recovery and purity were 61% and 83%, respectively. Finally, regeneration of the MW chromatographic column was studied to explore its recycling performance in application.