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Immunoassay is the most widely used detection technique in clinical testing. Compared with the traditional enzyme-linked immunoassay, the chemiluminescence immunoassay system based on carboxylated magnetic beads as the separation tool is more advantageous, which can rapidly separate proteins and achieve the purpose of quantitative detection of proteins. Separation tools in chemiluminescence immunoassay techniques are key and the focus of research. However, the domestic technology of preparing carboxylated magnetic beads is still immature, and the market is monopolized by imported products, which is not conducive to the development of domestic chemiluminescence immunoassay technology. Based on this, we propose a simple and convenient new method for the preparation of magnetic microbeads. Firstly, styrene-methyl methacrylate microspheres were polymerized by dispersion polymerization and hydrolyzed to form carboxylated microspheres, then carboxylated microspheres were introduced in the process of classical coprecipitation reaction to synthesize magnetic microbeads, and magnetic microbeads with different magnetic contents were prepared and characterized. The separation effect was then tested by a fully automated chemiluminescence immunoassay analyzer, and it was found that carboxylated magnetic beads with a magnetic content of 20% were the most effective in separating proteins, and the coefficient of variation was as low as 3.41%, with a stable and reproducible performance. The chemiluminescence immunoassay technique can separate proteins in a short period of time with a very small amount of carboxylated magnetic microbeads, which is fast and efficient and will help in the early diagnosis of diseases in healthcare facilities and may be a better point-of-care assay.Graphical abstract: Immunoassay is the most widely used detection technique in clinical testing. Compared with the traditional enzyme-linked immunoassay, the chemiluminescence immunoassay system based on carboxylated magnetic beads as the separation tool is more advantageous, which can rapidly separate proteins and achieve the purpose of quantitative detection of proteins. Separation tools in chemiluminescence immunoassay techniques are key and the focus of research. However, the domestic technology of preparing carboxylated magnetic beads is still immature, and the market is monopolized by imported products, which is not conducive to the development of domestic chemiluminescence immunoassay technology. Based on this, we propose a simple and convenient new method for the preparation of magnetic microbeads. Firstly, styrene-methyl methacrylate microspheres were polymerized by dispersion polymerization and hydrolyzed to form carboxylated microspheres, then carboxylated microspheres were introduced in the process of classical coprecipitation reaction to synthesize magnetic microbeads, and magnetic microbeads with different magnetic contents were prepared and characterized. The separation effect was then tested by a fully automated chemiluminescence immunoassay analyzer, and it was found that carboxylated magnetic beads with a magnetic content of 20% were the most effective in separating proteins, and the coefficient of variation was as low as 3.41%, with a stable and reproducible performance. The chemiluminescence immunoassay technique can separate proteins in a short period of time with a very small amount of carboxylated magnetic microbeads, which is fast and efficient and will help in the early diagnosis of diseases in healthcare facilities and may be a better point-of-care assay. [ABSTRACT FROM AUTHOR]