Monodispersed, readily-grafted, and biocompatible surface-enhanced Raman spectroscopic (SERS) tagging materials are developed; they are composed of bimetallic Au@Ag nanoparticles (NPs) for optical enhancement, a reporter molecule for spectroscopic signature, and a carbon shell for protection and bioconjugation. A controllable and convenient hydrothermal synthetic route is presented to synthesize the layer-by-layer triplex Au–Ag–C core–shell NPs, which can incorporate the Raman-active label 4-mercapto benzoic acid (4-MBA). The obtained gold seed–silver coated particles can be coated further with a thickness-controlled carbon shell to form colloidal carbon-encapsulated Aucore/Agshell spheres with a monodisperse size distribution. Furthermore, these SERS-active spheres demonstrated interesting properties as a novel Raman tag for quantitative immunoassays. The results suggest such SERS tags can be used for multiplex and ultrasensitive detection of biomolecules as well as nontoxic, in vivo molecular imaging of animal or plant tissues.