Superparamagnetic iron oxide nanoparticles with narrow size distributions were successfully prepared in large scale by a facile one-pot synthetic method in the presence of hydrophilic polymers, such as polyethylene glycol diacid (HOOC-PEG-COOH) and poly(acrylic acid) (PAA). The as-prepared products were investigated in detail by powder X-ray diffraction (XRD), thermogravimetric analyses (TGA), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), and vibrating sample magnetometer (VSM). The interaction between polymers and iron oxide nanoparticles was investigated using Fourier transform infrared spectrometry (FT-IR). The results show that polymers can be attached onto the surface of iron oxide nanoparticle by bridging coordination and monodentate fashion, respectively. The interaction affects iron oxide nanoparticle properties significantly, such as XRD diffraction intensity, hydrodynamic diameter, isoelectric point, and saturation magnetization. Furthermore, the results of in vitro experiments indicated that iron oxide-PEG-COOH nanoparticle is more cytotoxic than iron oxide-PAA nanoparticle due to different coordinating modes.