Iron oxide nanoparticles have been used for many years as clinical applications. We have developed a rapid immunoaffinity isolation method of CD4+T cells from a mixed cell population of human blood using iron oxide nanoparticles. Anti CD4-antibody has been attached to iron oxide nanoparticles after its surface modification. The antibody tagged iron oxide nanoparticle beads are simply incubated with the mixed cell population of human blood and CD4+T cells are purified using an external magnetic field. The purification level was checked by fluorescence microscopy and flow cytometry. The purified CD4+T cells were digested with trypsin with different time periods and the products were analyzed by MALDI-TOF mass spectrometry, without further fractionation or purification, to obtain its proteome pattern. A database search showed a number of peptide masses matched specific to T-cell peptide masses. These results indicate that iron oxide nanoparticles are useful for CD4+T cell purification, and mass spectrometry based proteolytic fingerprint is simple and swift for identifying putative surface biomarkers from the whole cell surfaces.