Leaf blight is a fungal disease that mainly affects the growth and development of leaves in plants. To investigate the molecular mechanisms of leaf blight defense in poplar, we performed RNA-Seq and enzyme activity assays on thePopulus simonii × Populus nigraleaves inoculated withAlternaria alternatefungus. Through weighted gene co-expression network analysis (WGCNA), we obtained co-expression gene modules significantly associated with SOD and POD activities, containing 183 and 275 genes, respectively. We then constructed a co-expression network of poplar genes related to leaf blight resistance based on weight values. Additionally, we identified hub transcription factors (TFs) and structural genes in the network. The network was dominated by 15 TFs, and four out of them, includingATWRKY75,ANAC062,ATMYB23andATEBP, had high connectivity in the network, which might play important functions in leaf blight defense. In addition, GO enrichment analysis revealed a total of 44 structural genes involved in biotic stress, resistance, cell wall and immune-related biological processes in the network. Among them, there were 16 highly linked structural genes in the central part, which may be directly involved in poplar resistance to leaf blight. The study explores key genes associated with leaf blight defense in poplar, which further gains an understanding of the molecular mechanisms of biotic stress response in plants.