Background: Green synthesis of nanoparticles has provided a cost-effective, environmentally friendly method, as well as raised safe strategies for the synthesis of nanomaterials. Methods: In this study, we investigated the synthesis of Selenium nanoparticles from the leaf aqueous extract of Moringa oleifera (MO-SeNPs) and studied the effect of MO-SeNPs and Moringa extract with various concentrations on the growth, active constituents, and antioxidant activity (scavenging DPPH) of Populus alba callus. MS medium supplemented with (2, 4-D at 2 mg/l + 0.5 mg/l BA) took the maximum callus induction percentage (100%), increasing the callus fresh weight, dry weight, and degree of callus formation compared with (2.0 mg/l 2,4-D + 0.5 mg/l Kin). Results: The highest values of callus fresh and dry weight were recorded on MS containing 20% MO-SeNPs. Regarding the active constituents, the GC-MS chromatogram revealed the presence of 23 major compounds identified in the chloroform extract. The phytoconstituents with maximum peak area in callus extract are Tetradecane, Hexadecen-1-ol, Trans-9, 5-Octadecene, (Benzene, 1- pentyloctyl), (Benzene, 1-butylnonyl), Cycloeicosane, and 10-Heneicosene in all treatments. Moringa extract and MO-SeNPs stimulate the increase of compounds in callus compared to control. In this regard, MO-SeNPs 5% led to a higher increase in the area percent of active substances compared to the treatment of Moringa extract and control. Concerning the antioxidant activity, the results indicated that Moringa extract and MO-SeNPs had a positive effect on scavenging DPPH compared with the control. The highest percentages of scavenging DPPH were recorded when Populus alba L. callus treated with MO-SeNPs 20%, 10%, and Moringa extract 20%, percentages reached (84.65, 75.35, and 71.94%), respectively. Conclusion : Green synthesis of MO-SeNPs improved secondary metabolites and antioxidant activity in Populus alba L. callus.