The assembly of metal nanoparticles (NPs) has attracted a great deal of attention recently because of their collective properties that could not be exhibited by individual NPs. Here a one-step approach was reported for the fabrication of spherical silver NP assemblies (AgNAs). The formation of AgNAs simply included the stirring of silver ammonia and 3,4-dihydroxy-l-phenylalanine (DOPA) in aqueous solution at room temperature, in which DOPA acted as a reductant for AgNPs first because of its reducing ability and then directed the assembly of AgNPs into AgNAs. The AgNAs exhibited hierarchical structure with controllable sizes ranging from 180 to 610 nm by adjusting the concentrations of reagents. The two individual components, AgNPs and polyDOPA, also allowed AgNAs with multiple functions as demonstrated in this study of durable catalytic activity, high SERS sensitivity, and good antioxidant properties. The thin polyDOPA layer coated on AgNAs further offered the opportunity to modify the surface of AgNAs. The results presented here may provide a green and facile approach to designing multifunctional NP assemblies.