Disclosed is a process of preparing magnetite nanoparticles, comprising the following steps: 1) preparing a ferric salt mixed system, wherein a soluble ferric salt is dissolved in glycol at ambient temperature, and then urea and polyethylene glycol are added and mixed homogeneously to obtain the trivalent iron salt mixed system, the mass ratio of glycol to the trivalent iron salt being 15:1 to 60:1, glycol to urea being 20:1 to 100:1, and glycol to polyethylene glycol being 20:1 to 100:1; 2) reacting, wherein the trivalent iron salt mixed system is transferred into a reaction autoclave, sealed and placed into a heating device to react at a temperature of 200 to 300° C. for 8 to 72 hours; and 3) washing, wherein after the reaction system is naturally cooled down to ambient temperature, the product is taken out, and washed with anhydrous ethanol and water in turn to obtain the magnetite nanoparticles. The soluble iron salt includes ferric chloride, ferric sulfate, ferric acetate and ferric nitrate. The obtained nanospheres exhibit a uniform distribution of the particle diameter with a good dispersity in water. The nanospheres have superparamagnetism, and their particle diameter can be controlled by varying the reaction time as desired.