The conjugates of magnetic beads coupled with an antibody can be trapped on the Au film firmly due to the magnetic force for the immunoassay of a surface plasmon resonance (SPR) biosensor. However, this approach exhibits significant limitations in robustness and sensitivity due to incomplete dissociation of magnetic beads from the Au film. The incorporation of a polyelectrolyte film on the Au surface can prevent the magnetic beads from the direct contact with the Au film. The layer-by-layer assembly of polyelectrolyte was used as spacer between the gold surface and the magnetic bead. Different layers of polyelectrolyte can be assembled onto the Au film based on an electrostatic force between polycations and polyanions. After the polyelectrolyte film was fabricated on the Au film, the deposition of the magnetic beads was maintained effectively on the film, which favors the sensitivity of the biosensor and the regeneration of the sensing membrane. When the polyelectrolyte layers of (PAH/PSS)(3) were constructed on the Au film, the SPR biosensor with magnetic beads exhibited a satisfactory response to human IgG in the concentration range from 0.25 to 30.00 microg mL(-1), and the determination limit obtained is eight times lower than that obtained with (PAH/PSS)(1) layer.