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This study aims to establish a highly sensitive and specific method for single cell screening and identification of KRAS+/epithelial cell adhesion molecules (EpCAMs)/vimentin+/pancreatic cancer circulating tumor cells (CTCs) and to explore the clinical implications in the diagnosis of pancreatic cancer. A single cell separation and identification system for pancreatic cancer CTCs was constructed based on the preparation of KRAS immunolipid magnetic spheres. The function of the system was evaluated, the physical parameters of the immunoliposomes (IMLs) were characterized, and the isolation effect of the IMLs on CTCs was studied and compared with EpCAM and vimentin phenotypic CTCs. CTCs in the peripheral blood of 50 patients with pancreatic cancer confirmed by preoperative CT diagnosis and postoperative pathology were isolated and identified. The isolated CTCs were stained with CK immune antibody, observed, and identified, the number of CTCs was counted, and the relationship between the CTC count and the clinical malignant grade markers and grade indicators was examined. The consistency of the KRAS mutation rate between CTCs and tissues was analyzed and compared. It was found that the circulating tumor cell sorting system with a high expression of KRAS-ML could effectively enrich the circulating tumor cells with a positive expression of KRAS in pancreatic cancer. The pancreatic cancer CTC sorting and single-cell gene detection system can be used as a new detection technology of peripheral blood CTCs in patients with pancreatic cancer. As a minimally invasive liquid biopsy method, this technology has significant clinical practice implications for pancreatic cancer detection and effectiveness assessment. [ABSTRACT FROM AUTHOR]