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Multiplexed assays are essential for the detection of biomarker panels. Differentiating signals from different biomarkers in a single test zone makes the detection more efficient. In this paper, a new method is designed for the synthesis of gap-enhanced nanoparticles (GeNPs) using Raman reporter molecules (RRM) and 6-amino-1-hexanethiol (6-AHT) as the spacer. The GeNPs show a nanometer-size gap, generate strong surface-enhanced Raman scattering (SERS) attributed to the gap, and exhibit discriminative spectral peaks. The strong Au-S bonds on both core and shell sides and the covalent bond between RRM and 6-AHT led to a stable structure, which ensured the stable SERS signal generation from the GeNPs. Using the GeNPs, a spectrally multiplexed assay for the detection of a biomarker panel is developed. The biomarker panel is composed of cardiac troponin I (cTnI), copeptin, and heart-type fatty acid-binding protein (h-FABP), which improves myocardial infarction (MI) diagnostic performance. A paper-based platform that is more amenable to point-of-care diagnostic analysis is used. The developed single biomarker assay achieves limits of detection of 0.01 ng mL