Orientedly bioconjugated core/shell Fe(3)O(4)@Au magnetic nanoparticles were synthesized for cell separation. The Fe(3)O(4)@Au magnetic nanoparticles were synthesized by reducing HAuCl(4) on the surfaces of Fe(3)O(4) nanoparticles, which were further characterized in detail by TEM, XRD and UV-vis spectra. Anti-CD3 monoclonal antibody was orientedly bioconjugated to the surface of Fe(3)O(4)@Au nanoparticles through affinity binding between the Fc portion of the antibody and protein A that covalently immobilized on the nanoparticles. The oriented immobilization method was performed to compare its efficiency for cell separation with the non-oriented one, in which the antibody was directly immobilized onto the carboxylated nanoparticle surface. Results showed that the orientedly bioconjugated Fe(3)O(4)@Au MNPs successfully pulled down CD3(+) T cells from the whole splenocytes with high efficiency of up to 98.4%, showing a more effective cell-capture nanostructure than that obtained by non-oriented strategy. This developed strategy for the synthesis and oriented bioconjugation of Fe(3)O(4)@Au MNPs provides an efficient tool for cell separation, and may be further applied to various fields of bioanalytical chemistry for diagnosis, affinity extraction and biosensor.