We describe the fabrication of plasmonic‐active nanostructured thin film substrate as a label‐free surface‐enhanced Raman scattering (SERS)‐based biosensor immobilized covalently with monoclonal HER‐II antibody (mAb) to detect overexpressed HER‐II as a biomarker in breast cancer serum (BCS). Oriented conjugation of mAb via hydrazone linkage to provide higher mAb accessibility was characterized by UV‐vis and reflective Fourier transform near‐infrared (FT‐NIR) spectroscopic techniques. The interaction of BCS with mAb was studied by FT‐NIR and nonresonant SERS at 637 nm. The results showed detection of glycoprotein content at different laser powers including a rise in amino acid and glycan content with varying results at higher power. With nonresonant SERS we observed nonlinear behavior of peak intensity. Analysis of variance was implemented to determine the effect of laser power which was found not to be a contributing factor. However, at the nanoscale, factors including the heating effect and aggregation of molecules can contribute to the nonlinearity of peak intensity.