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Magnetic nanoparticles have had a significant impact on a wide range of advanced applications in the academic and industrial fields. In particular, in nanomedicine, the nanoparticles require specific properties, including hydrophilic behavior, uniform and tunable dimensions, and good magnetic properties, which are still challenging to achieve by industrial-scale synthesis. Here, we report a gram-scale synthesis of hydrophilic magnetic nanoclusters based on a one-pot solvothermal system. Using this approach, we achieved the nanoclusters with controlled size composed of magnetite nanocrystals in close-packed superstructures that exhibited hydrophilicity, superparamagnetism, high magnetization, and colloidal stability. The proposed solvothermal method is found to be highly suitable for synthesizing industrial quantities (gram-per-batch level) of magnetic spheres with unchanged structural and magnetic properties. Furthermore, coating the magnetic spheres with an additional silica layer provided further stability and specific functionalities favorable for biological applications. Using in vitro and in vivo studies, we successfully demonstrated both positive and negative separation and the use of the magnetic nanoclusters as a theragnostic nanoprobe. This scalable synthetic procedure is expected to be highly suitable for widespread use in biomedical, energy storage, photonics, and catalysis fields, among others.