A dual-SERS encoding strategy was designed for in situ and in vivo evaluation of multiplex protein-specific glycosylation of tumors. The dual-SERS encoding strategy consisted of two pairs of dual gold nanoprobes with different diameters of 10 and 30 nm, which were encoded with four different and distinguishable Raman signal molecules. The 10 and 30 nm gold nanoprobes (Au10 and Au30 probes, respectively) were further modified with lectins and aptamers to recognize the target glycans and proteins, respectively. After sequential binding to the target glycans and proteins, the adjacent Au10 and Au30 probes could emit strong surface-enhanced Raman scattering (SERS) signals to indicate the multiplex protein-specific glycosylation information on cells and in vivo, which can reveal in situ the distribution differences of different tumor markers in the central and marginal regions of tumors. This strategy has been successfully applied for in situ imaging and evaluation of the MUC1 and EpCAM-specific Sia and Gal/GalNAc information on cell surfaces and tumor xenografted mice, providing a convenient and powerful tool to study protein-specific glycosylation-related physiological and pathological mechanisms.