Flavonoids are key secondary metabolites that are biologically active and perform diverse functions in plants such as stress defense against abiotic and biotic stress. In addition to its importance, no comprehensive information has been available about the secondary metabolic response of Populus tree, especially the genes that encode key enzymes involved in flavonoid biosynthesis under drought stress. In this study, the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the expression of flavonoid biosynthesis genes (PtPAL, Pt4-CL, PtCHS, PtFLS-1, PtF3H, PtDFR, and PtANS) gradually increased in the leaves of hybrid poplar (P. tremula × P. alba), corresponding to the drought stress duration. In addition, the activity and capacity of antioxidants have also increased, which is positively correlated with the increment of phenolic, flavonoid, anthocyanin, and carotenoid compounds under drought stress. As the drought stress prolonged, the level of reactive oxygen species such as hydrogen peroxide (H2O2) and singlet oxygen (O2−) too increased. The concentration of phytohormone salicylic acid (SA) also increased significantly in the stressed poplar leaves. Our research concluded that drought stress significantly induced the expression of flavonoid biosynthesis genes in hybrid poplar plants and enhanced the accumulation of phenolic and flavonoid compounds with resilient antioxidant activity.