A novel label-free amperometric immunosensor for the detection of rubella vaccine was developed by immobilizing anti-rubella serum on bilayer nano-Au/polymerized o-phenylenediamine film with electrodeposited Prussian Blue (PB) as an electrode transfer mediator on the platinum electrode. The redox reactions of PB as a probe on the platinum surface were blocked due to the binding of the antibody to the antigen, which was investigated by cyclic voltammetry. Therefore, the interaction of the antibody with various concentrations of antigen could be detected by measurements of amperometric response in PBS, and the amperometric response on the surface of the modified electrode was inversely proportional to the concentration of rubella vaccine in the sample. The immunosensor showed a specific response to rubella vaccine in the range 8.1×10−8–8.0×10−6 lgCCID50/ml (cell culture infectious dose) and a detection limit of 4.010−8 lgCCID50/ml at a signal-to-noise ratio of 3. To summarize, the present work provides a low-cost, fast response time, highly sensitive and easy-to-prepare method for the determination of antigen in biological products.