Objective: To explore the effect of peeling rate on the content of different nutrient active substances, antioxidant capacity, noodle quality and texture characteristics in Tibetan No. 27 highland barley flour. Methods: By controlling the peeling time, Tibetan No. 27 highland barley flour with different peeling rates could be obtained. The “National Food Safety Standard” and Megzyme kit were used to detect the content of fat, protein, minerals, dietary fiber and β-glucan and so on. Compared the effects of different peeling rates on the digestibility, antioxidant activity, noodle quality, and texture characteristics of Tibetan No. 27 highland barley flour. Results: ① The peeling rate of 4% had a good effect on retaining and enhancing the nutritional value of Tibetan No. 27 highland barley flour, which was suitable for making crispy food. ② The content of total sugar and β-glucan in the outer cortex of Tibetan No. 27 highland barley were less. ③ Non-essential amino acids were mainly concentrated in the embryo and endosperm of Tibetan No. 27 highland barley. ④ The insoluble dietary fiber of Tibetan No. 27 highland barley played a major role in starch hydrolysis and blood sugar control. ⑤ The total flavonoids were mainly concentrated in the outer cortex of Tibetan No. 27 highland barley, which played a major role in total antioxidant capacity and DPPH⁺ clearance. ⑥ Total phenols had a better scavenging effect on ABTS⁺ and O-₂. ⑦ Tibetan No. 27 highland barley flour milled with a peeling rate 8% was suitable for pressed noodles. Conclusion: The peeling rate had a greater impact on the nutrient and active substances of Tibetan No. 27 highland barley flour. When the peeling rate was 4%, the prepared highland barley flour had higher nutritional value and was suitable for crisp food. When the peeling rate was 8%, the prepared highland barley noodles tasted better.