The immunomagnetic beads coated with monoclonal antibody (McAb) against FITC were prepared and preliminarily applied. McAb against FITC was prepared by B lymphocyte hybridoma technique. The prepared ascites were preliminarily extracted with 50% ammonium sulfate and further purified with anion exchange resin DE52 to obtain McAb. Nano magnetic particles were prepared by oxidation co-precipitation method, while the magnetic microsphere with carboxyl group by emulsion polymerization method. McAb against FITC was linked onto the surface of magnetic microspheres with carbodiimide (EDAC) to prepare immunogmagnetic beads. Five hybridoma cell strains were obtained, of which 1F12 secreted the McAb with high titer, relative affinity and specificity. The purified McAb 1F12 reached a purity of 95%, with a protein content of 2.4 mg/mL, an ELISA titer of 106, a relative affinity of 0.2 mg/L, and showed specific binding to FITC-labeled BSA. The mean diameter and ferric content of prepared nano magnetic particles were 150 nm and 71.63% resp. However, the mean diameter and carboxyl group content of prepared magnetic microspheres with carboxy group were 210 nm and 2.15 mmol/g resp. Each gram of magnetic microspheres with carboxyl group was bound to about 12 mg of McAb against FITC, and the prepared immunomagnetic beads was bound to FITC-labeled protein effectively. The prepared immunomagnetic beads coated with McAb against FITC showed higher sensitivity than ELISA in determination of HBsAg, of which the detection limit was 0.1 ng/mL. Immunomagnetic beads coated with McAb against FITC were prepared successfully, which might be used for immunoassay.