A magnetic separation device for immunoassay using a two-sided plate for multi-well microplate separation via a flat side containing a magnetic block and a back side of magnetic microplate structure containing a matrix of permanent magnets. The present invention relates to a multi-purpose magnetic separation device that has two sides of magnetic separation function. The flat side of magnetic separation device includes magnetic block, metal or plastic sheet surface top and a pin to secure the assay plate. The back side of magnetic separation device is a multi-well structure that includes a plurality of permanent magnets placed in the spaces between the adjacent wells. There are different magnetic configurations for the multi-well structure, which ensures that each well is pulled by at least one strong permanent magnet.