Objective To explore the influence of the whole blood samples cold storage time on detecting serum hepatitis B virus DNA (HBV DNA) by real-time fluorescent quantitative PCR (FQ-PCR). Methods Whole blood samples of 150 patients with hepatitis B virus were collected in vacuum tubes containing coagulant. These samples were centrifuged in 1 hour and stored in cold storage (2~8℃). The serum HBV DNA levels in the samples were detected by FQ-PCR at 1st day and at 7th day. Results The HBV DNA load levels of 150 patients were 5.50±1.46 and 5.64±1.53 at 1st and 7th day respectively. These results showed an upward trend, but there was no significantly difference between two groups (P>0.05). Compared with the results of the first day, it had a quiet tolerance of (4.26±0.03) % at the 7th day, and among these 150 samples, more than 7.5% deviation was found in 15 samples. Conclusion On the premise of the quality control in the course of FQ-PCR, the influence of the whole blood samples’ cold storage time on detecting serum HBV DNA fits the requirements of ISO15189, which can meet the clinical needs. Inexact operation and methodological weaknesses are the main causes of deviations.