Functional polymeric microgels have great potential for biomedical applications owing to the outstanding advantages of their well-defined structures and tunable surface properties. However, traditional polymerization methods lack the efficient techniques needed for the fabrication of polymeric microgels. Herein, we have developed a novel technique, reflux-precipitation polymerization, for the preparation of a new type of monodisperse poly(ethylene glycol methacrylate phosphate) (PEGMP) microgel with a well-defined spherical structure and abundant phosphate groups. Furthermore, we have extended this technique to fabricate various monodisperse microgels baring different functional groups, including crosslinked PVIM, PHEMA and PVPBA microgels, as well as non-crosslinked poly(AAm-co-MAA) nanohydrogels, which could serve as promising candidates for specific protein enrichment and drug delivery systems. The morphologies and particle size distributions of the microgels were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), whereas the surface properties were measured via a zeta potential test. In addition, the use of the PEGMP microgels for the identification of phosphoproteins in both a protein mixture and drinking milk was investigated, and demonstrated high selectivity towards phosphoproteins.