This study aims to portray the characteristics of oxidative stress (OS) in cases of Necrotizing enterocolitis (NEC), identify the hub genes and associated mechanisms involved, and explore potential drugs for NEC. We performed a comprehensive analysis integrating bulk-RNA sequencing and single-cell RNA sequencing datasets, coupled with various techniques including differential analysis, gene set enrichment analysis, and immune infiltration analysis. We aimed to systematically elucidate the variations in functions related to OS among distinct cell populations within both NEC and non-NEC tissues. Additionally, we depicted the longitudinal changes in immune cells, with a particular focus on macrophages, throughout the progression of NEC. NEC mice model was established and RT-qPCR was performed to validate the expression of the hub genes. In total, 465 OS related genes were found, and 53 of them were significantly differentially expressed. These genes were mainly involved in several signaling pathways, such as TNF signaling pathway, IL-17 signaling pathway, FOXO signaling pathway, inflammatory bowel disease. The top 10 hub genes were , , , , , , , , and . Ten kinds of drug were discovered as the potential treatment for NEC. Four specific macrophages subtypes and relative function were identified in NEC. RT-qPCR and immunofluorescence staining confirmed the expression of the hub genes in NEC model. This investigation yielded innovative insights into the immune environment and therapeutic methodologies directed at oxidative stress in the pathogenesis of NEC.