Background and Aims Chronic skin ulcers are a significant disease affecting patients' daily lives and psychological well-being. Abnormalities in the cells and extracellular matrix within the tissue may disrupt the balance of the microenvironment, hindering the normal skin repair process and leading to delayed healing of the ulcer. There is currently a lack of research on the mechanisms underlying the development of chronic ulcers and their diagnostic biomarkers. Single-cell sequencing, a newly developed high-throughput sequencing method in recent years, uses gene sequencing at the single-cell resolution to precisely reveal disease mechanisms and has been applied in various diseases. This study used single-cell transcriptome sequencing (scRNA-Seq) to investigate the cellular heterogeneity in chronic skin ulcer tissue to elucidate the potential molecular mechanisms behind delayed healing and provide new insights for clinical treatment.Methods The scRNA-Seq technology was used to compare the differences in cell subpopulations and gene expression between chronic ulcer tissue and normal skin tissue. Single cells were sorted using a microfluidic platform, and cDNA libraries were constructed for subsequent differential gene analysis and functional enrichment analysis.Results scRNA-Seq analysis revealed significant immune-metabolic remodeling features in chronic ulcer tissue: the number of B cells, monocytes, and macrophages in ulcer tissue increased by 2.1 to 3.5 times compared to the normal tissue control. This was accompanied by widespread activation of collagen synthesis genes (COL1A1/COL3A1) and synergistic suppression of immune regulators (e.g., granzyme family GZMA/GZMB/H). Cross-cell subpopulation functional network analysis showed that hypoxia response mediated by the HIF-1 signaling pathway and PI3K/Akt pathway abnormalities formed a positive feedback loop, exacerbating the imbalance in the secretion of inflammatory factors (CXCL3/8, TGFBI) and compensatory upregulation of mitochondrial oxidative phosphorylation.Conclusion Chronic skin ulcers exhibit significant differences in cellular heterogeneity and gene expression, suggesting that chronic ulcers are not simply tissue defects but a complex pathological process dominated by chronic inflammation and immune dysregulation. The coordinated dysregulation of multiple cell subpopulations in the ulcer microenvironment, along with persistent inflammatory responses and metabolic abnormalities, is interconnected through the HIF-1/TNF/MAPK pathway network. Downregulation of granzyme gene family members and abnormal histone modifications may contribute to immune clearance defects, providing a theoretical basis for developing novel therapies targeting epigenetic regulation or mitochondrial function.