Immunoassays using nanomaterials have been rapidly developed for the analysis of multiple biomolecules. Here, highly sensitive and biocompatible surface enhanced Raman spectroscopy-active nanomaterials have been used for biomolecule analysis in order to overcome intrinsic problems of conventional immunoassays. Fluorescent surface-enhanced Raman spectroscopic dots (F-SERS dots) are composed of silver nanoparticle-embedded silica nanospheres, organic Raman tagging materials, and fluorescent dyes. We successfully applied the F-SERS dots for the detection of three cellular proteins simultaneously expressed in bronchioalveolar stem cells in murine lung, and further analyzed the relative expression ratios of each protein. Quantitative comparisons of multiple protein expression in tissue were first attempted using SERS-encoded nanoprobes. Our results suggested that immunoassays using F-SERS dots offered significant increases in sensitivity and selectivity for multiplex targeting, tracking, and imaging of cellular events in living organism. Such immunoassays may serve as the primary next-generation labeling technologies for the simultaneous analysis of multiple biomolecules.