Abstract: Response surface methodology was used to study the effects of germination time, pH value and sodium selenite concentration on organic selenium contents in highland barley seeds, and the optimal conditions were obtained. Under the optimal conditions, the contents of antioxidant substances and antioxidant abilities in highland barley seeds during germination were further analyzed. The results showed that the optimal conditions were as follows:Germination time 48.00 h, pH6.00 and sodium selenite concentration 10.00 mg/L. Under these conditions, the contents of organic selenium in highland barley seeds was 1.572 mg/kg DW, which was 17.46 times higher than that in raw material. Selenium-enriched germination could significantly increase the contents of total phenolics, total flavonols, organic selenium and ABTS⁺ radical scavenging ability of highland barley seeds (p<0.05). Although β-glucan contents and ferric reducing antioxidant power (FRAP) were decreased, the degradation of β-glucan contents was effectively delayed by selenium-enriched germination compared with the control group. Correlation analysis showed that total phenolics contents was highly positively correlated with organic selenium contents and ABTS⁺radical scavenging ability (p<0.01). Meanwhile, there were an extremely significant positive correlation relationship between β-glucan contents with ferric reducing antioxidant power (FRAP), and between organic selenium contents with ABTS⁺radical scavenging ability (p<0.01), while other indexes were negatively correlated or not correlated. This study showed that selenium enrichment treatment could increase the contents of antioxidant substances, effectively delay the degradation of β-glucan and enhance the antioxidant abilities in germinated highland barley seeds.