The optimum conditions for synthesizing leucine (Leu)-binding nano-sized magnetite (NSM) particles by a two-step transformation (TST) process were studied. The formation and magnetization of as-synthesized NSM particles were investigated through variation of the acidity, the type of surface modifier, and the nature of the acid for pH adjustment. With increased acidity, the saturation magnetization of the NSM particles decreased, but the amount of Leu coated on the surface of NSM particles increased. After the influence of hydroxyl (OH.) groups on the surface of NSM particles was removed by using the dicarboxyl anion (C2O42.) as a ligand in the first step, Leu was successfully bound with NSM particles in the second step. However, when polyethylene glycol (PEG) was used as a surface modifier, it was difficult to form the Leu-to-NSM particle complex. In terms of the acid used to modify pH, H2SO4 was slightly less effective than HCl in achieving saturation magnetization because the coordination for SO42. anions is stronger than that of Cl. anions. The preparation of other amino acid-binding NSM particles can be optimized in an analogous manner.