Barley sprout (Hordeum vulgare L.) is believed to have antioxidant, lipid metabolic, anti-cancer, and anti-fatty liver formation effects. However, the benefits of barley sprout to improve blood flow have not been studied yet. The purpose of this study was to investigate the blood flow improvement effect of barley sprout water extract in vitro on serotonin release, thromboxane A2 release, platelet aggregation, activated partial thromboplastin time, and prothrombin time-induced microvesicle generation. Serotonin secretion was significantly reduced 400 μg/mL of barley sprout water extract as compared with 0 μg/mL. Thromboxane A2 secretion from the phospholipid membrane was significantly reduced at 400 μg/mL as compared with 0 μg/mL. Platelet aggregation was significantly reduced at 400 μg/mL as compared with 0 μg/mL. Activated partial thromboplastin time in plasma was significantly increased at 400 μg/mL as compared with 0 μg/mL. Prothrombin time in plasma was not significantly different. In conclusion, these results suggest that barley sprout water extract at 400 μg/mL ameliorated blood flow.