Multiplex microsphere-based immunofluorescence assay is a reliable, accurate, and highly sensitive method for the detection of various biomolecules. However, for the moment, the wide application of the method in clinical practice is prevented by the high cost of reagents for analysis-magnetic spectrally encoded microspheres. Therefore, an urgent task is the development of new methods for the synthesis of microspheres with the required properties. The aim of this study was the creation of new magnetic fluorescent microspheres suitable for use in multiplex immunoassay. Samples of magnetic fluorescent polystyrene microspheres were synthesized by dispersion polymerization and two-stage swelling methods. Experimental studies of geometric parameters, fluorescence, magnetic properties of the synthesized microspheres have been carried out. The results of the studies have shown that microspheres synthesized by dispersion polymerization are promising for the use in immunofluorescence analysis. The obtained results can be used for the development of new diagnostic multiplex test systems based on spectrally encoded microspheres.