Surface-imprinted microspheres were controllably synthesized using ionic liquid-functionalized microspheres with a highly cross-linked raspberry-like structure as the matrix via surface molecular self-assembly and precipitation polymerization in aqueous media at room temperature. An immunostimulating hexapeptide from human (IHH) with medical properties was chosen as a template molecule in the preparation of different molecularly imprinted microspheres (MIMs). The experiment process was tracked and the as-prepared microspheres were well characterized. Results reveal that the adsorption capacity and selective recognition of MIMs have a direct relationship with the properties of the functional chain of the ionic liquid-functionalized microspheres. Moreover, MIMs that have both high adsorption capacity and good selective recognition were used in competitive rebinding tests and analysis of urine samples, which demonstrated their potential use for IHH enrichment and in real samples.