Iris pseudacorus L. is an important endangered plant and is commonly cultivated for its aesthetic and medicinal value. Optimization of in vitro culture conditions, callus development, somatic embryogenesis, and plant regeneration constitutes the critical and initial step for successful propagation. This study investigated the effects of various concentrations(low to high)of 2,4-Dand kinetin on callus formation parameters and secondary metabolite production. Conducted as a factorial experiment with a completely randomized design, it included15treatments(plant growth regulators) under different light conditions (light and darkness). Results indicated that darkness had a more pronounced positive effect on callus development than light. The highest total phenol content(47.95 mg(100g)-1)was found in the treatment of 7.5mgL-12,4-D+0kinetin. Additionally, the treatment of 2.5 mgL-1 2,4-D+0.5 mgL-1 kinetin yielded the highest flavonoid content(72.08mgg-1), while10mgL-12,4-D+ 1mgL-1kinetin produced the lowest. Light conditions favoured greater flavonoid production compared to darkness. Treatment interactions did not significantly affect anthocyanin production. The highest amount of anthocyanin was produced (0.0804 µmol(gFW)-1) in the treatment of 0 mgL-1 of 2,4-D and 1 mgL-1 of kinetin. About embryogenesis rate, the highest rate observed in callus subcultures in MS medium supplemented with1mgL-1 kinetin+1 mgL-1IBA. The embryogenesis rate positively correlated with increased phenol content, this study demonstrated successful somatic embryogenesis, callus formation, and enhanced secondary metabolite production through optimized hormonal compounds at novel concentrations in Yellow Flag, which had not been tested until now. The methods presented in this research are very valuable for increasing the production of secondary metabolites of this important medicinal and ornamental plant in the pharmaceutical industry.