In this study, biocatalytic synthesis of iron oxide nanoparticles was carried out by urea hydrolysis catalyzed by enzyme urease and with the addition of iron (II) chloride. Optimization of the process using a statistical method of Design of Experiments was performed in order to determine optimal conditions for the synthesis of nanoparticles with the smallest size and the highest concentration. Influence of temperature change (25°C – 40°C) and concentration of urease (0,5 – 3 mg mL^-1) with constant concentration of urea and iron (II) chloride was examined. Kinetics of enzyme urease was studied in the reaction of urea hydrolysis by measuring the concentration of ammonium ions at different concentrations of urea with constant concentration of urease. Kinetics of the reaction was described with Michaelis – Menten kinetic model. Influence of iron ions on enzymatic activity was examined by measuring the specific activity of urease at different concentrations of iron (II) chloride and at constant concentrations of urea and urease. It has been found that iron ions inhibit urease by non-competitive inhibition and that the inhibition is increased with temperature increase Assumed mathematical model of the process was validated in a batch reactor. All experiments were carried out in redistilled water at temperatures T = 25 °C, T = 32,5 °C and T = 40 °C. Synthesized iron oxide nanoparticles were characterized with a scanning electron microscope with energy dispersive spectroscopy and nanoparticle tracking analysis.