This study was aimed to investigate the effects of different Lactobacillus and fermentation conditions on the enrichment of polyphenols in whole grain black barley， in which No.1 Lactobacillus hilgardii (DSMZ 20051)， No.2 Lactobacillus fermentum (DSMZ 20052)， No.3 Lactobacillus diolivorans (DSMZ 14421) and No.4 strains Lactobacillus kisonensis (JCM15041) were used in the fermentation. The results showed that different fermentation strains had significant effects on polyphenols enrichment of black barley (P<0.05). The polyphenols enrichment effect of No.4 strains Lactobacillus kisonensis(JCM15041) was better than other three strains. The optimal fermentation conditions were established as follows via single factor and orthogonal experiments: solid-liquid ratio 1∶5， inoculum volume of No.4 strains 3%， fermentation time 24 h， fermentation temperature 30 ℃. Under these optimized conditions， the polyphenols content reached (0.88±0.016) mg/g， which was 1.05 times higher than that of unfermented black barley. Some of polyphenols compounds such as bergenin， cinnamic acid， maltol， catechin， genkwanin， trans-coumaric acid， caffeic acid， anthocyanidin， and ferulic acid were identified in the extract of black barley fermentation broth by using the targeted metabolomics analysis of UPLC-Q-TOF-MSE， which were 3.82， 7.95， 1.38， 0.21， 0.43， 1.40， 0.37， 0.36 times and 0.33 times higher than that of unfermented black barley(P<0.05). Additionally， syringic acid and veratric acid were only detected in fermented black barley. In conclusion， fermentation of black barley with Lactobacillus kisonensis (JCM15041) could effectively enrich polyphenols， which can be used for the preparation of functional whole grain food material.