Surface-enhanced Raman scattering (SERS) is an ultrasensitive optical technique that is critical for protein detection and essential for identifying protein structure and concentrations in various biomedical and diagnostic applications. However, achieving highly sensitive and reproducible SERS signals for label-free proteins remains challenging due to their weak Raman signals and structural complexity. In this study, silver nanomushroom arrays (Ag NMAs) as SERS substrates were readily prepared and surface-engineered using a facile template-assisted micro- and nanofabrication approach. The surface of the substrate exhibits nanoscale roughness, long-range order, and hydrophilicity, enabling rapid and uniform dispersion of protein molecules. These molecules are anchored through Ag-S bonds, resulting in ultrasensitive Raman signals driven by strong electromagnetic enhancement effects. The highly ordered array structure improves signal repeatability, achieving a relative standard deviation of as low as 4.32%. Additionally, utilizing the silicon characteristic peak of the SERS substrate as an internal standard significantly reduces measurement errors, allowing for reliable and precise quantitative detection of protein molecules, with a linear correlation coefficient (