Objective To analyze the results of nonrepetitive reactivity of HBV nucleic acid detection in blood donors. Methods 1 200 blood samples from our station were confirmed by nonrepetitive reaction, supplemented with the “two to half” test of hepatitis B, and some patients with single nucleic acid detection were followed up. Results One hundred and fifty samples (12.50%) were reactive in single nucleic acid detection in 1 200 blood samples. After the virus nucleic acid detection, 58 samples were reactive (38.67%), 92 were nonreactive (61.33%). 18 donors were actually recalled and drew blood, completed two tracking. The first tracking time was 60-125 days, the second time interval was 160-356 days. In the first tracking 8 cases had HBV DNA reaction (+) , which still had reaction in the second tracking; 7 HBV DNA positive donors had HBC antibody (+).The proportion of HBV-DNA positive with 10-17 value in mucleic acid reaction test(37.25%) was higher than that with value below 10(4.17%,P＜0.05). Conclusion Some donors with single nucleic acid detection reaction have a certain risk of transmission of HBV, most of them had hidden infection of HBV. At this time, we should pay attention to the blood transfusion and avoid the single nucleic acid reactive donors.