We have reported on a novel enzyme immunoassay method for the detection of protein using biocatalytic silver nanoparticles as an enhanced substrate based on surface-enhanced Raman scattering (SERS). First, ascorbic acid was converted from ascorbic acid 2-phosphate by alkaline phosphatase immobilized on polystyrene microwells after a typical sandwich immunoreaction. Then Ag(I) ions were reduced to silver nanoparticles by the obtained ascorbic acid, which would result in a SERS signal when Raman dyes were absorbed. Using human IgG as a model protein, a wide linear dynamic range (1 to 100 ng ml(-1)) was reached with a low detection limit (0.02 ng ml(-1)) under the optimized assay conditions. Moreover, the production of an enhanced substrate was chosen as the signaling element in this method, which demonstrates a new way for SERS-based quantitative detection. These results suggest that the application of SERS enhanced by biocatalytic production of metal nanopaticles holds a promising potential for a sensitive immunoassay.