In order to explore the mechanism of processing methods affecting protein nutrition in whole grain foods, cooked whole-grain highland barley products (rice) and flour-based products (noodles) were selected as the research objects, with raw highland barley flour used as the control. The effects and underlying mechanisms of cooking and milling on protein nutrition in whole grain highland barley were evaluated based on the nutrient composition of indigestible residues, as well as the structure and amino acid composition of digestion-resistant proteins. The results showed that the digestibility of both rice and noodles was significantly reduced compared to raw flour (P<0.01) and the gastrointestinal digestibility of noodles was increased compared to rice. Cooking promoted the interaction between major nutrients and inhibited the digestion of protein, among which starch had the strongest inhibitory effect on the digestion of protein in highland barley, followed by dietary fiber. Milling improved the digestibility of protein because it promoted the digestion of starch. Both cooking and milling treatments enhanced the indigestibility of the alcohol soluble protein subunits (D, B, B3), partial alcohol soluble protein subunits γ-3 and the albumin subunit Serpin z-type, partly because cooking and milling promoted the formation of disulfide bonds (raw flour 0.91 μmol/g; rice 1.22 μmol/g; noodles 1.68 μmol/g). In addition, the contents of hydrophobic amino acids in indigestible residues of highland barley were significantly different (P<0.05). The contents of rice were the highest, followed by noodles, and raw flour was the lowest. It showed that the indigestibility of protein was positively related to the contents of hydrophobic amino acids. After cooking and simulated digestion, digestible indispensable amino acid score (DIAAS) was reduced, the first limiting amino acid was Lys which remained the same, the second limiting amino acid was leucine (Leu) for rice and isoleucine (Ile) for noodles. The results of the study are helpful to understand the interaction between macromolecular substances in complex food systems, clarify the influence mechanism of cooking and milling processing on protein nutrition, help to understand the bioavailability of highland barley protein in different processed products, and provide a new theoretical basis for protein nutrition evaluation of whole grain foods.