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An impedance immunosensor was developed recently for the rapid detection of a H5 subtype avian influenza virus (AIV). The important step was to have a magnetic nanobead separator (MNS) to separate and concentrate the streptavidin-coated magnetic nanobeads which were captured with the avian influenza virus H5N1. This paper describes a compact and portable magnetic nanobead separator designed for the rapid detection of an avian influenza virus and the series of experiments for confirming the separation efficiency. These ideas came from several tries of different MNS separator designs and experiments. There were six separation holes constructed by tile-shaped NdFeBs in this MNS. It was built up of a cylinder shell with 6 cylinder containers, pairs of tile-shaped magnets, a piece of metal magnetizer, and 6 separation holes. The design was aided by Pro/Engineer software to simulate and analyze. From the measurements, the maximum magnetic induction intensity of each separation area (hole) reached 1166.2mT and its maximum grads reached 152.7T/m. Also, the ingenious configuration of MNS could be suitable for other requirements by minor amendment. The experiments on separating H5N1 virus and E. coli O157:H7 were conducted to confirm the separation efficiency of the MNS with the nanobeads in different sizes. The results showed that the MNS could separate 30, 100, and 180nm nanobeads entirely, while the separation duration was no less than 60min, 60, and 40s respectively, and the separation efficiency was higher than 96.5% under the controlled experimental condition, without any residual bead in supernatant liquid. The experiments for separation efficiency of the MNS included: a multi-point magnetic induction intensity measurement, a preliminary separation observation, a transmission electron microscope observation, a Dot-ELISA experiment, and an enumeration by a plate count experiment. All the repeatable experimental data showed that the MNS system was stable and efficient. The relative standard deviation (RSD) of the magnetic induction intensity measurement was around 3%. [ABSTRACT FROM AUTHOR]