Hydrophilic 2,3-dimercaptosuccinnic acid (HOOC-CH(SH)-CH(SH)-COOH, DMSA) coated monodisperse magnetic nanoparticles (Fe3O4) were dispersed in water, RPMI-1640 with 10% (v/v) fetal calf serum, RPMI-1640, PBS and MES, respectively, to investigate their stability under biologically relevant conditions. The Photon Correlation Spectroscopy (PCS) results showed that DMSA-Fe3O4 nanoparticles existed as aggregate under biological conditions. UV-vis, MRI and AFM results indicated that DMSA-Fe3O4 nanoparticles dispersed in RPMI-1640, PBS and MES presented poor stability, whereas those dispersed in RPMI-1640 with fetal calf serum exhibited excellent stability, which was due to their adsorption from fetal calf serum, as confirmed by zeta potential and IR results. Additionally, in vitro cell experiments showed that DMSA-Fe3O4 nanoparticles with adsorption from fetal calf serum had higher intracellular uptake than those without adsorption from serum, indicating that fetal calf serum could play a great role in intracellular uptake of nanoparticles.