A simple and economical method to isolate whey protein from fresh raw milk is developed by serial defatting, casein eliminating, lactose removing, and separating by gel filtration chromatography. Four major whey components, including immunoglobulin (Ig), bovine serum albumin (BSA), beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-Lac), and a non-protein of low molecular mass ( approximately 1.7 kDa) but strong absorbance at 280 nm, are detected simultaneously. The small non-protein molecule is rich in aromatic amino acids and thiol groups as supported by the structural characterization with near infrared Fourier transform Raman spectroscopy (FT-Raman). FT-Raman results show that the secondary structure of Ig is dominated by anti-parallel beta-pleated sheet; BSA is mainly in alpha-helix; both beta-form and unordered structure are important in beta-Lg; while alpha-Lac is mostly in alpha-helix coupling with random coil. Differences in the Raman profile for each whey component reflect their intrinsic compositional differences and distinct spatial arrangement. The S-S linkages diverging around 510-540 cm(-1) indicate that the conformation of disulfide bonds in each whey components is different, which may be responsible for their diversified behaviors in solubility, rheological and functional properties.