In the present study, an accurate and reproducible method for quantifying cell-free DNA (cfDNA) in human blood was established and tested for its ability to predict gastric cancer in patients. Using 'Alu81-qPCR' to amplify 81-bp Alu DNA sequences, we first estimated the amount of cfDNA in the serum or plasma of 130 patients with gastric cancer to identify which source of cfDNA is more suitable for the biomarker screening of these patients. The results of Alu81-qPCR revealed that the amount of cfDNA in the plasma was low compared with that in the serum, but was found at similar levels among the samples, indicating that the plasma may be a more suitable source of cfDNA for biomarker screening. For the 54 patients with gastric cancer and the 59 age-matched healthy controls, the mean levels of plasma cfDNA were 2.4-fold higher in the patient group compared with the control group, indicating that plasma cfDNA levels may be useful for predicting patients with gastric cancer. The results of our study suggest that Alu81-qPCR is a more reliable method than other techniques, such as the PicoGreen assay, for quantifying cfDNA in human blood, demonstrating the potential to complement current diagnostic procedures for the management of gastric cancer patients.