Magnetophoresis is a motion of a magnetic or magnetizable particle induced by an inhomogeneous magnetic field in a fluid. Magnetophoretic immunoseparation, using micro- or nano-sized magnetic particles often modified by monoclonal or polyclonal antibodies for specific separation of biological or chemical targets, has shown a great potential in continuous-flow separation of cells and bacteria in clinical and biomedical fields. In this paper, the basic knowledge, key design considerations and recent developments on magnetophoretic immuno-separation of biological targets were reviewed.