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• The two-dimensional SERS substrate can be used for qualitative and quantitative detection of IL-6 in serum samples. • Au@PB@Au NPs can be used as SERS tags to quantitatively detect IL-6, which only have SERS signal in biological immersion area. • The limit of detection of our proposed detection method is 0.5 pg/mL for IL-6. We successfully prepared a two-dimensional surface enhanced Raman spectroscopy(SERS) substrate for the trace detection of Interleukin-6(IL-6) in plasma. Glycoproteins in serum were captured by a two-dimensional silver plate modified with 4-mercaptobenzoic acid (4-MPBA). And then, the Au@Au core-shell nanoprobes modified with IL-6 antibody accurately recognized IL-6 glycoprotein. The Au@Au NPs were embedded with Prussian Blue (PB) molecule, which only peaked at 2172 cm−1 in the biological silence zone. The calibration model of 4-MPBA and PB double signal molecules can avoid the interference of other glycoprotein macromolecules in the environment, thus realizing the quantitative detection of IL-6. The results showed that the higher the IL-6 content, the stronger the Raman signal. And the detection limit of IL-6 in serum can reach 5 pg/mL, the applicable range is 0.5–50000 pg/mL (R2 = 0.9928). Therefore, this method can be used as a multifunctional SERS activity technology, which can be used to detect IL-6 ultra-sensitively and quantitatively. [ABSTRACT FROM AUTHOR]