Spherical Polyelectrolyte Brushes (SPBs) are nanoparticles formed by densely-grafted polyelectrolyte chains on the surface of colloidal particles. Because of the Donnan effect and electrostatic interaction, SPBs can confine a large number of counterions in aqueous solution, especially at relatively low concentrations, which make them effective nanoreactors for the preparation of metal or metal compound catalysts in situ. Organic–inorganic hybrid functional nanomaterials including hollow nanoparticles can be generated using SPBs as templates. In addition, SPBs are ideal nanocarriers in the separation and selective immobilizing of proteins as the interactions between SPBs and proteins can be tuned by changing the pH value, ionic strength, and protein concentration. In this chapter, recent advances in SPBs are systematically reviewed. We conclude that SPBs as functional nanoparticles show great potential for applications in catalysis, multi-functional nanomaterials preparation, controlled release, enzyme immobilization, and protein separation.