Surface-functionalized magnetic nanoparticles were prepared by a facile one-pot solvothermal method in ethylene glycol solution. Zeta value, size, and magnetic properties could be well tuned by introducing different functional group molecules. Characterizations, including transmission electronic microscopy, scanning electronic microscopy, thermogravimetric analysis, x-ray powder diffraction and vibrating sample magnetometer, and Fourier transform infrared spectrophotometer demonstrated the efficiency of this simple and general synthesis strategy. The hydrophilic magnetic nanoparticles with various surface functional groups and zeta values were evidenced as excellent candidates for bioseparation by extracting DNA molecules from a model mixture of cell fractures.