Magnetite (Fe3O4) particles were prepared for biomedical application through three different routes. Each route had the difference in the selection of pH controlling agents. Ammonia, Urea and combined use of ammonia and urea were tested in the synthesis through co-precipitation. With the combined use of urea and ammonia, the size of particles could be reduced as small as 10 nm while effectively suppressing the agglomeration among particles. The surface charge measured at physiological condition explained the good dispersion behavior of nano-magnetite particles. The superparamagnetism observed in nano magnetite particles was expected to be useful for biomedical application in the respect of magnetic localization and dispersion.