Secondary structural, superparamagnetic Fe3O4 microparticles with an average diameter of 280 nm have been successfully synthesized by using a one-step hydrothermal method. The size of the primary nanograins has been controlled from 5.9 to 21.5 nm by varying the sodium acrylate/sodium acetate weight ratios. The magnetic properties of the Fe3O4 microparticles have been characterized at room temperature, whereas the saturation magnetization values of the Fe3O4 microparticles increase with increasing grain sizes. Magnetic resonance imaging reveals that Fe3O4 microparticle with larger grain size yields higher molar T2 relaxation rate. A plausible growth mechanism of the particles is proposed, and the role of sodium acrylate and sodium acetate for tuning the grain size of the particles has been discussed. Additionally, the size of the secondary structural Fe3O4 particles can also be continuously controlled from 6 to 170 nm by varying the volume ratio of ethylene glycol/diethylene glycol in a bisolvent system. The described method presents the synthesis of secondary structural nanomaterials with tunable sizes, grain sizes, and different magnetic responses.