Barley tea (Mugicha), commonly consumed in Japan and other East Asian countries, is prepared by decocting roasted barley (Hordeum vulgare) seed with hot or cold water. Although barley tea is commonly consumed, studies on its health benefits are limited, especially regarding its bioactivity against thrombosis. During the evaluation of functional foods and drinks, barley tea extract was found to exhibit potential fibrinolysis-enhancing activity induced by urokinase. Therefore, the aim of this study was to explore the application of barley tea as a functional food and conduct a preliminary investigation to reveal the effects of barley tea on thrombosis. Hot water extract of roasted barley was treated with pancreatin and separated via various techniques using macroporous resin and silica gel and tangential flow filtration with an ultra-filtration membrane. The low-molecular-weight fraction of the roasted barley tea extract was found to possess activity. We further purified the extract and found that the activity of each fraction decreased. Thus, the different fractions of the roasted barley extract may not exhibit the activity individually, instead additive or synergistic effects of multiple components may occur. The results suggest the potential use of barley extract as a functional food to prevent thrombosis.