This paper reports a low-cost method of continuous size-dependent sorting of magnetic nanoparticles in polymer-based microfluidic devices by magnetic force. A neodymium permanent magnet was used to generate a magnetic field perpendicular to the fluid flow direction. Firstly, FeNi3 magnetic nanoparticles were chemically synthesized with diameter ranges from 80 nm to 200 nm; then, the solution of magnetic nanoparticles and a buffer were passed through the microchannel in laminar flow; the magnetic nanoparticles were deflected from the flow direction under the applied magnetic field. Nanoparticles in the microchannel will move towards the direction of high-gradient magnetic fields, and the degree of deflection depends on their sizes; therefore, magnetic nanoparticles of different sizes can be separated and finally collected from different output ports. The proposed method offers a rapid and continuous approach of preparing magnetic nanoparticles with a narrow size distribution from an arbitrary particle size distribution. The proposed new method has many potential applications in bioanalysis field since magnetic nanoparticles are commonly used as solid support for biological entities such as DNA, RNA, virus, and protein. Other than the size sorting application of magnetic nanoparticles, this approach could also be used for the size sorting and separation of naturally magnetic cells, including blood cells and magnetotactic bacteria.