The purpose of this study was to explore the reliability of cell-free DNA (cfDNA) as an indicator for screening patients who benefit from neoadjuvant chemotherapy (NACT) in advanced gastric cancer (GC). 70 GC patients with TNM (Tumor, Lymph Node, Metastasis) stage II-III were enrolled. Plasma specimens of GC patients before and after NACT and of 50 healthy volunteers were collected. The concentration and integrity of cfDNA were detected by qRT-PCR. cfDNA concentration and integrity of different groups were analyzed to explore its relationship with clinical characteristics of gastric cancer patients. ROC (Receiver operating characteristic) curve was established to compare the cfDNA sensitivity and specificity with cancer antigen 724 (CA724), carcinoembryonic antigen (CEA), cancer antigen 199 (CA199) and alpha-fetoprotein (AFP). Factors affecting the prognosis of advanced GC patients were analyzed by COX univariate/multivariate analysis. The result showed that plasma cfDNA concentration and integrity of advanced GC patients before NACT were significantly higher than those of normal people. After receiving NACT, cfDNA concentration and integrity were significantly decreased (p < 0.05). There was a significant correlation between cfDNA concentration and TNM stage (p < 0.05). The values of area under curve (AUC) of ROC curve for cfDNA concentration and integrity were greater than those of CEA, CA724, CA199 and AFP. COX analysis showed that the tumor differentiation degree and cfDNA concentration were independent risk factors for the advanced GC patients prognosis. In conclusion, cfDNA can be used to predict the prognosis of advanced GC patients, and as a reliable indicator to evaluate for further NACT in advanced GC patients.