The phytochemical profiles of fermented barley with <i>Lactiplantibacillus plantarum</i> dy-1 (LFBE) and its antioxidant activity were investigated. Nine components including cinnamaldehyde, homovanillic acid, coumalic acid, quercetin, docosahexaenoic acid methyl ester, gallic acid, cafestol, 6-hydroxycaproic acid, and indole-3-lactic acid were identified by HPLC-MS/MS and evaluated as potential antioxidants in LFBE. DPPH and ABTS scavenging ability, superoxide dismutase (SOD) activity, reactive oxygen species (ROS) level in HepG2 cells, and the cellular antioxidant activity (CAA) were assessed. The results indicated that the nine components showed good radical scavenging capacities, most of them had the abilities of reducing ROS level and promoting SOD activity. In CAA tests, coumalic acid exhibited the highest value (11.51 μmol of QE/100 g), while gallic acid showed the lowest value (3.47 μmol of QE/100 g), which was not consistent with the trend of other evaluations. This study provided a basis for understanding the effects of fermentation on bioactivity of barley.