Conjugation and characterization of poly-ethylene–glycol (PEG)-functionalized gold nanourchin (GNU) with breast cancer biomarker HER-II monoclonal antibody (mAb) (i.e., anti-HER-II) for selective targeting are described. After the functionalization of GNU with PEG, the surface plasmon resonance (SPR) peak was red-shifted, indicating the increase in the hydrodynamic size of the GNU. The Fourier-transform near-infrared spectroscopy (FT-NIR) second derivative result of GNU-PEG provided overtone and combination bands of fundamental vibrational modes of protein molecular structures between 4000 and 7500 cm−1. This mainly included C–H combination and CH2 bonds, O–H first stretch overtones, the C–H first stretch overtone, and the CH2 combination first overtone. The UV–Vis absorbance showed a strong absorption of light at 227 and 275 nm corresponding to tyrosine peaks. The fluorescence emission peak at 315 nm corresponds to Stokes shift when excited by 280 nm corresponding to tyrosine in the mAb, and the peak at 497 nm likely corresponds to alanine. After conjugation of GNU-PEG with mAb, the FT-NIR indicated the bands corresponding to NH2 combination and amino acids, first overtone symmetric and antisymmetric OH stretching, C–H combination, and the second overtones and combination modes. Surface-enhanced Raman scattering (SERS) provided useful information on the molecular structure and composition of the sample within 300–3500 cm−1. The intensity behavior of SERS signals exhibited a statistical nature due to Brownian fluctuating movement. In addition, the intensity and number of SERS lines varied with the laser power. The dominant peaks were corresponding to histidine, tyrosine, tryptophan, phenylalanine, and C–H, N–H, C–N, and O–H bonds.