The molecular structure of many proteins contains disulfide bonds between their cysteine residues. In this work we demonstrate the utilization of the disulfide bond structure of proteins for their label-free determination by surface-enhanced Raman spectroscopy (SERS). The new approach for label-free SERS detection of proteins is demonstrated for human insulin. The protein was selectively extracted from spiked plasma samples using target-specific functionalized nanomaterial. Enzyme-linked immune assay (ELISA) was used to detect insulin in the blood plasma and cross-validate the SERS method. The disulfide bonds in the molecular structure of the protein were chemically reduced and used for their chemisorption onto the gold-coated copper oxide substrate in a unified orientation at a very short distance from the hotspots. The oriented chemisorption of the protein caused significant enhancement to the signal intensity of its Raman vibration modes. This is attributed to the strong short-range electromagnetic and chemical enhancement effects that are experienced by the immobilized protein. Using this approach, label-free and reproducible SERS detection of insulin, down to 10 zM (relative standard deviation [RSD] = 5.52%), was achieved. Sixty-five percent of proteins contain disulfide bonds in their molecular structure. Therefore, the new label-free SERS detection method has strong potential for the determination of ultralow concentrations of proteins at pathology labs and in biology research.