Magnetic iron oxide nanoparticles (MIONPs) were synthesized in a FeCl₂-NaNO₃-NaOH aqueous system under various initial Fe(2+)/NO⁻₃ molar ratios (α) and Fe(2+)/OH- molar ratios (β) in order to clarify the effects of the initial molar ratio of reactants on the reaction mechanism. The Fe(2+)/NO⁻₃ /OH(-) molar ratio of 3 : 1 : 5 led to the formation of magnetic nanoparticles mainly composed of magnetite (Fe₃O₄) and maghemite (γ-Fe₂O₃). The 36 nm sized γ-Fe₂O₃ and 413 nm sized Fe₃O₄ were obtained by changing the order in which NaNO₃ was added to a NaOH solution. The in vitro heat generations of the resulting MIONPs in an agar phantom were measured under an alternating magnetic field (100 kHz, 23.9 kA/m). The temperature rise (ΔT) of the agar phantom for the 36 nm sized γ-Fe₂O₃ was 55°C in the first 140 s, with a concentration of 58 mg Fe/mL. Our results showed that it is possible to prepare MIONPs with high heating efficiencies under optimal conditions using the present method.