Sub-100 nm hollow gold nanoparticle superstructures were prepared in a direct one-pot reaction. A gold-binding peptide conjugate, C-6-AA-PEPAu (PEPAu = AYSSGAPPMPPF), was constructed and used to direct the simultaneous synthesis and assembly of gold nanoparticles. Transmission electron microscopy and electron tomography revealed that the superstructures are uniform and consist of monodisperse gold nanoparticles arranged into a spherical monolayer shell.