In order to clarify the difference of nutritive composition of each milling layer of highland barley rice, the nutrients and functional composition in different milling layer of powder were compared. The results showed that there were significant differences in the content of nutrients in different milling layers of highland barley (P<0.05). The contents of crude fiber, arabinoxylan, Ca, K, Mn and Na were the highest in the first milling bran. While, the contents of fat, ash, crude protein, GABA, Fe and Mg in bran were highest in the second milling bran. The content of total starch, Cu and Zn in the barley rice was highest and the color was most white, while the content of β-glucan in the highland barley raw grain was the highest. Heavy metals Hg, Cd, As and Pb were not detected in different milling layer powders. The content of glutamic acid was highest and the content of methionine was lowest in the powders of different milling layers. The amino acid composition of highland barley raw grain was closest to the human amino acid pattern. The quality of amino acids in the first milling bran was relatively better, and the nutritional value score of protein in the second milling bran was highest. Different types of polyphenols, flavonoids and total phenols were significantly different in different milling powders (P<0.05). Among them, the content of free phenol and total phenol in the third milling bran was highest, while the content of bound phenol, free flavonoid and bound flavonoid were all highest in the first milling bran. This study was aimed to provide theoretical reference for the deep processing and utilization of different milling layer powders of highland barley.