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The polypeptide sequence MS14 (MHGKTQATSGTIQS) was used to explore a new method for biomimetic preparation of gold nanoparticles and their aggregates. Self-congregation of gold nanoparticles into aggregates in MS14 aqueous solution and self-assembly of gold crystallites onto the designed complex of MS14-PET film [protonated poly(ethylene terephthalate)] proved the specific gold-binding characteristic of the single-copy peptide MS14 in vitro. In aqueous solution MS14 could recover Au(III) to Au(0), tested by means of TEM, EDX and XPS. Further research suggested that the pH of the solution and the concentration of Au(III) influenced the morphology and size of the gold nanoparticles formed. In addition, extra reducing agent, sodium citrate, was introduced into the HAuCl4-MS14 system and uniformly dispersed nanoparticles under neutral condition were obtained. Finally, we discuss the possible mechanism of this biomimetic synthesis.