Fluorescent polystyrene microbeads have been widely used in the biomedical and chemical fields. One of the key applications is the suspension array technology (SAT) based on fluorescence-encoded microbeads (FEMs). However, SAT is limited by the preparation of FEMs. Herein, a new preparation method of FEMs with large encoding capacities was designed, and the application of FEMs in SAT was estimated. By taking advantage of the functional monomers, the microbeads were reconstructed. First, the particle size of the polymer microbeads in this study is uniform, and the CV was 5.3%. Secondly, the surface functionalization of the polymer microbeads was realized. The content of carboxylic acid on the surface was 0.56 mmol g⁻¹, which is much higher than that available commercially. Furthermore, the concentrated fluorescence signal was proved by flow cytometry, which also indicated the successful modification of the surface functions groups. Then, the reconstitution of the polymer microbeads improved its compatibility with dyes. At the same concentration, the maximum fluorescence intensity of FEMs was approximately 5-fold as high as that of fluorescent polystyrene microbeads (FMs). Finally, the antigen was captured by FEMs combined with the antibody, indicating that FEMs can be applied to SAT. The great protein binding and weak non-specific adsorption ability of FEMs make it attractive to multiple detections in practical diagnosis.