Objective: To analyze the inconsistency between enzyme-linked immunosorbent assay (ELISA) HBsAg-negative and nucleic acid amplification assay (NAT) HBV DNA positive in HBV screening of unpaid blood donors in Changzhou, and further ensure the safety of blood transfusion. Methods: The qualified blood donor samples tested by two different ELISA reagents were further tested by the Roche or Kehua nucleic acid detection system for detecting HBV DNA, HCV RNA and HIV RNA in 6 mixed blood samples, and the positive POOLs were separated and detected. The specimens with positive segregation were tested by chemiluminescence for detection of 5 markers of hepatitis B, and the blood with 5 negative results was traced. Results: A total of 48 635 ELISA-negative blood donor samples tested for 2 times were mixed with 11 016 POOLs. The number of positive POOLs was 66 in the mixed test, and the number of POOLs that were split into HBV DNA positive was 40. No HCV RNA and HIV RNA were detected. The total effective resolution rate of NAT is 60.61%, and the positive rate of specimens detected by NAT is 0.08%. For the above-mentioned HBV DNA-positive blood, the five markers of hepatitis B were all negative in 7 cases by chemiluminescence; 6 cases of anti-HBs+, 6 cases of anti-HBs+/anti-HBc+, 4 cases of anti-HBs+/anti-HBe+, 7 cases of anti-HBc+/anti-HBe+, and 10 cases of anti-HBc+. Four negative cases were traced, and HBsAg in all these four turned from negative to positive. Conclusion: NAT can screen HBV DNA-positive specimens in ELISA-negative specimens, reduce the incidence of window period hepatitis B infection and occult hepatitis B infection, further ensure blood safety. The investigation shows that ELISA HBsAg-negative/NAT HBV DNA-positive blood donors were mainly occult hepatitis B virus infection, which was the main risk of residual blood transfusion.