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A simple, rapid, and sensitive method named critical desiccation state Raman spectroscopy (CDSRS) has been developed for detecting native and glycosylated protein. Raman spectroscopy was performed within 1 min and without background subtraction. Results showed that Raman intensity of 1 μL of low concentration dispersions of biomacromolecule analytes remarkably increased from weak to high signal-to-noise ratio during the progressive desiccation process, and high-quality CDSRS spectra of biomacromolecule analytes could easily be obtained under the critical desiccation state, especially those such as zein and whey protein isolate-dextran conjugate which were difficult and even impossible to detect from their solid powders or low concentration dispersions by conventional Raman spectroscopy. Therefore, CDSRS would provide an opportunity to obtain high-quality Raman spectra from biomacromolecule analytes, which would be beneficial to elucidate the relationship between secondary structures and functional properties of protein and to control the formation of food hydrocolloids with desirable physicochemical properties. [ABSTRACT FROM AUTHOR]