Immunoassays are analytical methods using antibody-specific reactions to analyze samples. Due to recent developments in antibody technology, the scope of potential samples has expanded to not only proteins, but also low molecular-weight compounds, carbohydrates, lipids, and microorganisms. Immunoassays have the advantage of being highly sensitive, capable of detecting small amounts, and thus have potential for application in biosensors. Immunoassays using magnetic beads have been developed and can be converted to more diverse platforms than the existing limited well plate-based assay. Furthermore, magnetic bead immunoassays detect analytical samples more quickly, and are becoming one of the most suitable immunoassay tools applicable to biosensors. However, their development requires optimization for the improvement of detection ability for specific samples. Therefore, we propose a guideline for solving detection problems occurring in magnetic bead immunoassay optimization processes. It is aimed to be a good reference, enabling researchers performing such optimization more quickly and efficiently.