As a novel immunoassay measurement, surface-enhanced Raman spectroscopy (SERS) was applied to multiplex labelled-immunoassay. Reporter molecules (thiophenol, bipyridine derivates, cyanopyridine derivates) were attached to different immuno-gold colloids, respectively. By means of the specific adsorption between antigen and antibody, a sandwich structure was constructed, in which labelled immuno-gold colloids and antigen covalently bound to substrate specifically capture different antibodies. SERS has the advantages of narrow band width, high resolution and low detection limit, which can distinguish the characteristic SER spectra of the reporter molecules, then the presence of multifarious antigens was established, and multi-analyte was detected successfully. Also the non-specific adsorption in multiplex labelled-immunoassay and adsorption of cyanopyridine derivates were discussed.