Magnetic separation of organic compounds, proteins, nucleic acids and other biomolecules, and cells from complex reaction mixtures is becoming the most suitable solution for large production in bioindustrial purification and extraction processes. Optimal magnetic properties can be achieved by the use of metals. However, they are extremely sensitive to oxidation and degradation under atmospheric conditions. In this work Ni nanoparticles are synthesised by conventional solution reduction process with the addition of a non-ionic surfactant as a surface agent. The nanoparticles were surfacted in citric acid and then coated with silica to form single core Ni nanobeads. A magnetophoresis study at different magnetic field gradients and at the different steps of synthesis route was performed using Horizontal Low Gradient Magnetic Field (HLGMF) systems. The reversible aggregation times are reduced to a few seconds, allowing a very fast separation process.Research highlights► Monodispersed single core Ni–silica core–shell structures were prepared. ► Control of Ni nanoparticles size was achieved using a non-ionic surfactant. ► Magnetophoresis at different magnetic field gradients was monitored. ► Magnetophoresis at different steps of synthesis route was performed. ► Attractive magnetic interactions overcome electrostatic repulsions.