Highly monodispersed magnetite nanoparticles (MNPs) were successfully prepared by means of a sonochemistry-assisted co-precipitation method in the presence of L-serine (L-ser). In order to understand the effect of the L-serine on the development of nanoparticles, various concentrations (0, 1, 3 and 5 mmol) of L-ser were employed. In this study, a conventional co-precipitation method was also performed to prepare MNPs (denoted as MNP-1) as a comparison. The MNPs prepared via sonochemistry-assisted co-precipitation method (MNP-2) have better crystallinity and morphological uniformity than MNP-1, which were observed by XRD and TEM measurements, respectively. Synergistically, the introduction of L-ser as a capping agent during the ultrasound irradiation significantly improved the particle size monodispersity and colloidal stability of MNP-2. In addition, the magnetic sensitivity of MNP-2 (Ms = 57.12 emu g−1) was not altered significantly even with the addition of 5 mmol of L-ser (Ms = 48.34 emu g−1). This investigation shows that the L-ser acts not only as a capping agent to improve the colloidal stability of nanoparticles but also as a particle size controlling agent.