This research explored the protective impact of bamboo leaf flavonoids (BLF) in diquat (DQ) stressed broilers; providing insight of antioxidant, immune response and intestinal barrier function. This experiment consisted of two parts. In the first, 240 chicks were allotted to 2 groups with 8 replicates and 15 chicks per replicate. Treatments consisted of a basic feed (control group, CON) and the basic feed plus 1000 mg/kg BLF (BLF group, BLF) for 28 d, respectively. Then, following the conclusion of the first part, 16 healthy broilers were selected from the CON group and the BLF group. They formed the second part of the experiment, and were allotted to 4 treatments with 8 broilers each: CON-no stress (CON-NS) group, CON-DQ group, BLF-NS group and the BLF-DQ group. Broilers were separately injected intraperitoneally with DQ solution at 40 mg/kg body weight or the same dose of phosphate buffer saline. The results revealed adding BLF to diet reduced the ratio of feed to weight gain of broilers compared to the basic feed group ( = 0.021). In comparison to the CON-NS group, BLF improved the levels of serum and jejunal mucosa total antioxidant capacity, immunoglobulin M, serum catalase, immunoglobulin A, interleukin 10, jejunal mucosa interleukin 4, cecal butyric acid, valeric acid, isobutyric acid, isovaleric acid, upregulated zonula occludens-1 (), occludin () and claudin-1 () expressions, and reduced the levels of jejunal mucosa malondialdehyde (MDA), interleukin 1β, interleukin 6 and serum diamine oxidase ( < 0.05). Diquat stress elevated the contents of serum MDA, D-lactate, jejunal mucosa tumor necrosis factor α, reactive oxygen species and relative abundance, downregulated , and expressions, and reduced Sobs, Chao and Ace indices ( < 0.05). Compared with CON-NS group, the concentration of isovaleric acid in the BLF-DQ group was higher ( < 0.05). In conclusion, by establishing a DQ stress injury model, it was elucidated that BLF may enhance antioxidant capacity, strengthen immunity, regulate volatile fatty acid contents, improve intestinal morphology, microbiota and other intestinal barrier functions, so as to mitigate the injury induced by oxidative stress in broilers.