Abstract: To modify highland barley protein by low temperature plasma technology, and to explore the effects of different treatment conditions on the structural and functional properties of highland barley protein. Scanning electron microscopy (SEM) was used to observe changes in the microstructure of highland barley protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to study changes in its primary structure. Fourier-transform infrared spectroscopy and fluorescence spectroscopy were utilized to analyze changes in its secondary and tertiary structures. Additionally, the impact of cold plasma on the basic functional properties of barley protein was analyzed. The results showed that with the increased treatment time, the surface morphology of the samples was disrupted, resulting in increased pore size and quantity. The primary structure of the protein remained unchanged after cold plasma treatment, while the β-sheet content in the secondary structure significantly increased (P<0.05), and the α-helix and random coil content significantly decreased (P<0.05). Moreover, the tertiary structure analysis revealed a significant increase in hydrophobicity (P<0.05) and a decrease in fluorescence intensity. Compared to untreated samples, after 40 min of cold plasma treatment, the solubility, water-holding capacity, emulsifying properties, and foaming properties of highland barley protein reached optimal levels. In conclusion, cold plasma treatment would improve the functional properties of highland barley protein by modifying its structural characteristics, offering important guidance for the utilization of highland barley protein and barley resources.