Because of their properties, nanoparticles of calcium carbonate have found application in medicine, textiles, food and packaging industries, which is why the development of ecologically, energetically and economically more acceptable methods of their synthesis is necessary. In this work, the optimization of biocatalytic synthesis of calcium carbonate nanoparticles by enzyme urease was carried out by using the statistical methods of Design of Experiments. The kinetics of urease were studied in the reaction of urea hydrolysis and the influence of calcium ions on the enzyme activity was determined. The reactions were carried out in redistilled water at a temperature of T = 25 °C, T = 32.5 °C and T = 40 °C. For the optimisation of the nanoparticle synthesis process, the input variables such as the temperature (25 ‒ 40 °C) and urease concentration (0.1 – 2 mg mL^-1) were varied within the certain ranges with constant concentrations of urea and calcium chloride. Reaction kinetics was experimentally determined using the initial reaction rate method at different concentrations of urea and with constant concentration of the enzyme. Kinetics of the reaction was described with Michaelis – Menten kinetic model. Influence of calcium ions on the enzyme activity was tested by measuring the specific activity of urease as a function of various concentrations of calcium chloride with a constant concentration of urea and urease. It was found that calcium ions inhibit urease by non – competitive inhibition. The assumed mathematical model of the process was validated in the batch reactor with constant mixing on the thermostatic shaker. Synthesized nanoparticles of calcium carbonate were analyzed by a scanning electron microscope with energy dispersive spectroscopy and nanoparticle tracking analysis.