The objectives of this study were to evaluate total phenolic, total flavonoid content and antioxidant activities of different extraction from three kinds of highland barley. The results showed that 95% ethanol and 60%acetone were better to extract phenolic compounds than other solvents. Total phenolic and total flavonoid contents, antioxidant activities of Zangqing 2000 using different solvents were higher than that of Xunhua and Shangri-la highland barley. Meanwhile, the highest total phenolic content of Zangqing 2000 was 211.92 mg GAE/100 g DW in 60% acetone, the highest total flavonoid content of Zangqing 2000 was 60.11 mg RT/100 g DW in 60% ethanol, the highest ABTS scavenging ability and total antioxidant activity of Zangqing 2000 were 1.85 and 9.28 mmol TEAC/100 g DW, respectively. Furthermore, there was a significant correlation between total phenolic content and antioxidant methods. FRAP method was significantly correlated to DPPH and ABTS methods. These results suggested that highland barley was rich in phenolic content and considered as a good natural antioxidant resource.