Silicon nanowires (SiNWs) offer promising inorganic nanostructures for biomedical application. Here, we report the development of a novel SiNW array designed for isolating primary CD4(+) T lymphocytes from the heterogeneous mixture of cell populations. Our system employed the specific high-affinity binding features of streptavidin (STR)-functionalized SiNW with biotin-labeled CD4(+) T lymphocytes. Fabricated SiNW arrays easily separated the CD4(+) T lymphocytes from the mouse whole splenocytes with over approximately 88% purity and demonstrated tight attachment to CD4(+) T lymphocytes by scanning electron microscopy. Thus, our STR-SiNW arrays provide a potential tool for specific cell separation and further present a possibility to be applied to the other area of biomedical applications.