Aim. In this study, the effects of temperature, Haucl4 concentration, and mixing ratio of the culture supernatant to Haucl4 on nanoparticles size were investigated. The study of these factors is important for achieving optimal and good conditions to produce au nanoparticles at a scale larger than experimental scale. Methods. In this work, au nanoparticles are synthesized by culture supernatants of E. coli. Muller-hinton broth (mhb) was prepared, sterilized and inoculated with a fresh growth of E. coli. After incubation the supernatant was used for subsequent experiments. Results. In order to identify and analyze nanoparticles, uv-vis spectroscopy, transmission electron microscopy (tern), and dynamic light scattering (dls) were used. In this research, it was found that initial concentration of Haucl4 plays a key role in formation of nanoparticles. In addition the results suggested that the best performance of nanoparticles synthesis was at 5 degrees C and mixing ratio 1 mL supernatant to 100 mL Haucl4 solutions. Conclusion. In this research, au nanoparticles synthesis using E.coli (dh5 alpha) in different conditions was studied. By achieving the best temperature, concentration and mixing ratio of culture supernatant to Haucl4, it was obtained suitable conditions for optimal production of nanoparticles.