Fibroblast activation protein (FAP) is an important antigen in the tumor microenvironment, which plays a crucial role in promoting extracellular matrix remodeling and tumor cell metastasis. A circulating form of soluble FAP has also been identified in the serum, becoming a biomarker for pan-cancer diagnosis and prognosis. However, the current peptide substrate-based enzymatic activity detection or antibody-dependent detection methods have been hindered by insufficient selectivity and complex operations, so it is valuable to develop effective nucleic acid aptamers as FAP affinity ligands. In order to deeply explore the biomimetic recognition technology, this study proposed an elaborate aptamer screening strategy for targeting the protein characteristic structure. Taking the glycosylation of the FAP protein as a target, four FAP-specific aptamers with high performance were successfully generated. Further, using the champion aptamer as a recognition tool and combining it with ultrasensitive detection technology-surface enhanced Raman scattering (SERS), a novel dual aptamer-based sandwich sensor was constructed for the rapid determination of FAP. Due to the dual-specific recognition of the orthogonal aptamer pair, the sandwich method obviously improved the selectivity to FAP protein, with a maximum cross-reactivity of less than 8% and a quantitation limit of 100 pg/mL. It was conveniently applied in high-sensitive and high-selective detection of serum FAP in cancer patient samples. Therefore, the research of this study not only opens new access for the selection of antiglycan aptamers but also boosts the application of the FAP aptamer as a recognition tool in cancer diagnostics.