The assembly of aqueous gold nanoparticles on the surface of polyurethane (PU) spheres leading to [gold nanoparticle shell]-[polyurethane core] structures is demonstrated. The assembly of gold nanoparticles on the polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles. Such direct assembly obviates the need to perform additional surface modification of the polymer microspheres, which is an important step in other polymer-based core-shell structure protocols. The nanogold-PU material is then conjugated with the enzyme pepsin, leading to the formation of a new class of biocatalyst. In relation to the free enzyme in solution, the new bioconjugate material exhibited a slightly higher biocatalytic activity and significantly enhanced pH and temperature stability. The use of gold nanoparticle-labeled polymer microspheres in pepsin bioconjugation enables easy separation from the reaction medium and reuse of the bioconjugate over six reaction cycles.