Background <p>To systematically investigate the dynamic changes in peripheral immune cell composition and function under different clinical phenotypes, detect important immune subpopulations and regulatory networks that cause drug-resistant epilepsy (DRE), and offer new theoretical and translational insights into immune classification, early intervention, and targeted therapy in pediatric DRE.</p> Methods <p>Peripheral blood mononuclear cells (PBMCs) from three DRE cases, two medication-effective cases (ME), and two healthy control cases (HC) were subjected to single-cell RNA sequencing (ScRNA-seq) analysis. The results showed variations in the proportions and gene expression profiles of immune cell types among the three groups. Differential gene trend analysis was conducted to screen signaling pathways and core genes associated with DRE. Monocytes, B cells, and T cells underwent further analysis, which identified cell subpopulations strongly related to DRE and changes in DRE-associated pathways. Finally, intercellular communication analysis was conducted.</p> Results <p>MBNL1, FOS, VPS13B, Rap1, and cAMP pathways related to DRE, as well as SKAP1, RAP1B, BRAF, and FOS genes were identified. Further in-depth analysis identified the VCAN⁺Mono subpopulation, as well as changes in DRE related pathways in T cells and B cells. Compared with the ME group, CD4⁺Tcm increased, and CD8⁺Tem decreased in patients with DRE. Finally, the DRE group exhibited enhanced pro-inflammatory properties of VCAN⁺Mono, increased exhaustion signaling in CD8⁺Tem, and reduced ability to adhere to and locate target cells. The small sample size may have affected statistical power, rendering some findings unreliable.</p> Conclusions <p>This is the first study to use scRNA-seq analysis on PBMCs from children with DRE, ME, and HC. The findings identified important genes and pathways that may be associated with DRE. It identified a VCAN⁺Mono subpopulation that is strongly associated with DRE and has pro-inflammatory properties. The results provide novel insights into the immune regulatory mechanisms of DRE and offer theoretical justification for the development of novel biomarkers and potential targets.</p>