<p>Ultraviolet-B (UV-B) is widely used in promoting the secondary metabolism and increasing the yields of bioactive and antioxidant materials in economical plants. Few studies have investigated the influence of UV-B combined with 6-benzyl aminopurine (6-BA) on the secondary metabolites and antioxidant capacities of <i>Scutellaria baicalensis</i> callus. This study aimed to investigate the effect of UV-B radiation on the antioxidant activity and the accumulation of secondary metabolites in <i>Scutellaria baicalensis in vitro</i> callus cultured at various 6-BA concentrations. The vigorous calli were incubated on MS medium augmented with different concentrations of 6-BA (0, 1, 2, and 3 mg·L^− 1) for 20 d and they were treated by UV-B light for 2&#xa0;h and harvested. Then, the malondialdehyde (MDA) content, content of secondary metabolites, antioxidant capacity were measured from the callus samples. The UV-B radiation significantly increased the callus lipid peroxidation, total phenolic and flavonoid contents, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) activity at low 6-BA concentrations (0, 1 mg·L^− 1), while they did not change significantly at high 6-BA concentrations (2, 3 mg·L^− 1). However, the UV-B radiation caused significant changes in SOD activity and contents of individual flavonoids (baicalin, scutellarin, wogonoside, baicalein, and wogonin) at all 6-BA concentrations tested. In conclusion, the UV-B radiation promoted the antioxidant activity and secondary metabolites accumulation in <i>S. baicalensis</i> callus, and their change was highly dependent on the 6-BA concentration added to the medium.</p>