A phage display-based bifunctional display system was developed for simple and sensitive immunoassay. The resulting bifunctional phage could simultaneously display a few single-chain variable fragment (ScFv) and many copies of the gold-binding peptide on its surface, thereby mediating antigen recognition and signal amplification. As a demonstration study, it was possible for bifunctional phage-based immunoassay to identify Bacillus anthracis spores from other Bacillus strains with detection sensitivity 10-fold higher than that of conventional phage enzyme-linked immunosorbent assay (ELISA). This protocol may be applied to build other bifunctional phage clones for broad applications (e.g., immunoassay kits, affinity biosensors, biorecognition assays).