Abstract: Single factor experiment and an orthogonal array design were used in combination to optimize the extraction conditions of polyphenols from barley Lactobacillus fermented solution ( BLFS) . High performance liquid chromatography ( HPLC) was employed to identify the chemical composition of polyphenols.Antioxidant activities of the extracted polyphenols were also measured in this study.The results showed that under the optimum conditions: ethanol concentration 60%, solid-toliquid ratio 1∶ 50 g/m L, extraction temperature 60 ℃, and extraction time 60 min, the extraction yield of BLFS polyphenols was ( 18.09 ± 0.25) mg/g.The results of chromatograph analysis with HPLC showed significant increase of gallic acid, coumaric acid, ferulic acid and rutin contents in barley after lactobacillus fermentation ( p < 0.05) . On the contray, vanillic acid and p-coumaric acid contents reduced dramatically ( p < 0.05) . In BLFS polyphenols, main components including rutin, vanillic acid, ferulic acid, coumaric acid, gallic acid, protocatechuic acid and p-coumaric acid, their contents were 35.08, 21.28, 19.38, 11.36, 6.80, 2.99 μg/g and 0.83 μg/g respectively. The polyphenols extraction from BLFS and barley displayed excellent antioxidant activities with half maximal effective concentration ( EC50) values of 0.66 and 0.87, 0.88 and 0.96, 0.76 and0.89 mg/m L for scavenging hydroxyl, DPPH radicals and reducing capacity, respectively. Antioxidant activities of BLFS polyphenols extraction were higher than these of barley polyphenols extraction, showed that barley increased antioxidant activity by Lactobacillus Fermentation.