The magnetic enzyme-linked immunoassay (MEIA) based on magnetic nanoparticles as the solid phase was reported in this work. Magnetic nanoparticles (MNPs) have represented perfectly suitable materials for a variety of biomedical and biotechnological applications. Therefore, we used MEIA based on magnetic nanoparticles to provide a screening method for fast analysis of serum Golgi protein 73 (GP73) in hepatocellular carcinoma (HCC) and healthy subjects and comparison was made with the enzyme-linked immunosorbent assay (ELISA) method. Several relevant conditions, including the concentration of anti-GP73 monoclonal antibody and HRP-anti-human GP73 monoclonal antibody, amount of immunomagnetic beads, and the incubation time, were determined and optimized. Finally, the MEIA was successfully established and validated by 79 HCC and 64 healthy subjects. The results showed this method achieved a detection limit of 0.78 ng/mL, which was more sensitive than ELISA. Furthermore, the sensitivity and specificity of the MEIA were 78.43% and 91.47%, respectively, which were higher than ELISA. The MEIA based on MNPs proved to be simple, sensitive, specific and time-saving, therefore holds great potential for development of a commercial kit in the future.