Nowadays, with the growth and development of industries, the requirement for steel and other iron products is increasing. Significant amounts of sludge and dust are produced every day in the steel companies. The presence of heavy metals, as well as the high content of iron in these resources, becomes the possibility of using these materials as an environmental and economic necessity. In this research, the iron-rich source of convertor sludge and dust of Isfahan Steel Company has been used as a secondary source of iron for the production of magnetite nanoparticles biologically. Iron solution for the biosynthesis of nanoparticles was prepared from leaching of sludge and iron dust as trivalent iron and sponge iron produced by Mobarakeh Steel Company of Isfahan as divalent iron. Ammonia solution produced by Bacillus subtilis was used to synthesize nanoparticles. The use of sugar beet vinasse as a source of sugar to bacteria instead of the chemical culture medium is appropriate environmentally and economically. The synthesis of nanoparticles was performed by adding dropwise solution of the iron solution with a ratio of Fe3+/Fe2+ = 2.45 to ammonia solution at 80 °C. Synthesized nanoparticles were examined by XRD, TEM, VSM, and FTIR analysis. The results of the XRD pattern detection proved the magnetite nature of the nanoparticles produced. According to the results of FTIR analysis, the surface of the nanoparticles is covered by biosurfactants, which prevents the accumulation of nanoparticles and caused to smaller dimensions of the nanoparticles. Based on TEM analysis, the size of nanoparticles produced is in the range of 11.6±5.03 nm. The saturation magnetization of nanoparticles was measured by VSM analysis, 58/91 emu / g and superparamagnetic nature of nanoparticles was confirmed. Finally, it can be said that biologically produced magnetite nanoparticles have many advantages over chemical methods such as more stability of produced nanoparticles, more regular shapes, no environmental pollution, high magneticity, and inexpensive culture medium.