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Background: In our previous study, a novel and sensitive method for quantifying circulating cell-free DNA (CFD) in human blood was established and tested for its ability to predict gastric cancer (GC) in patients.In the present work, we first used the branched DNA (bDNA)-based Alu assay to measure CFD levels, and then investigate CFD expression in the sera of GC patients in an attempt to explore the clinical significance of CFD in improving the early differential diagnosis of GC and monitoring GC progression.Methods: Serum samples were collected randomly from 124 unselected GC patients, 32 patients with gastric benign disease (BGD), and 92 healthy controls.Alu-based CFD concentrations were detected by branch DNA (bDNA).Concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 72-4(CA72-4), carbohydrate antigen 50(CA50) were detected by ARCHITECT assay.Results: There was a significant difference in CFD concentrations between GC patients, BGD patients and healthy controls (P<0.05).A significant correlation was discovered between CFD and CEA (r=-0.197, P<0.05) or CA50 (r=0.206, P<0.05), but there was no significant correlation between CFD and CA199 (r=-0.058, P >0.05) or CA724 (r=0.016, P>0.05).In addition, CFD concentrations in stage I GC patients were significantly higher than BGD patients and healthy controls (P<0.05), but for conventional biomarkers except CA72-4, there were no significant difference in CEA, CA19-9 and CA50 between the three groups (P>0.05).Our analysis showed that CFD was more sensitive than CEA, CA19-9, CA72-4 or CA50 in early differential diagnosis of GC.Conelusions: Compared with CEA, CA19-9, CA72-4 and CA50, CFD may prove to be a better biomarker for the early differential diagnosis of GC, thus providing a potentially specific tool for aided diagnosis of GC, and a sensitive biomarker for monitoring progression and predicting prognosis of GC.