Preparation of gold nanoparticles with a narrow size distribution has enormous importance in nanotechnology. Methanobactin (Mb) is a copper-binding small peptide that appears to function as an agent for copper sequestration and uptake in methanotrophs. Mb can also bind and catalytically reduce Au (III) to Au (0). In this study, we demonstrate a facile Mb-mediated one-step synthetic route to prepare monodispersed gold nanoparticles. Continuous reduction of Au (III) by Mb can be achieved by using hydroquinone as the reducing agent. The gold nanoparticles have been characterized by UV-visible spectroscopy. The formation and the surface plasmon resonance properties of the gold nanoparticles are highly dependent on the ratio of Au (III) to Mb in solution. X-ray photoelectron spectroscopy (XPS), fluorescence spectra and Fourier transform-infrared spectroscopy (FT-IR) spectra suggest that Mb molecules catalytically reduce Au (III) to Au (0) with the concomitant production of gold nanoparticles, and then, Mb statically adsorbed onto the surface of gold nanoparticles to form an Mb-gold nanoparticles assembly. This avoids secondary nucleation. The formed gold nanoparticles have been demonstrated to be monodispersed and uniform by transmission electron microscopy (TEM) images. Analysis of these particles shows an average size of 14.9 nm with a standard deviation of 1.1 nm. The gold nanoparticles are extremely stable and can resist aggregation, even after several months.