【Objective】 To study the detection performance of HBsAg single-ELISA-reactive samples of blood donors. 【Methods】 Two kinds of ELISA reagents from different manufacturers (named as reagents A and B) were used for HBsAg screening. A total of 276 samples, from January 2017 to May 2021, with HBsAg single-ELISA-reactive results were collected for further nucleic acid detection technology (NAT) and chemiluminescence immunoassay (CLIA) testing, to undergo HBV-DNA and five hepatitis B tests, respectively. The relationship between HBsAg single-ELISA-reactivity, NAT and CLIA was statistically analyzed. 【Results】 Among the 276 HBsAg single-ELISA-reactive samples, 14 were NAT reactive, with the positive rate of 5.07% (14/276). Fisher′s exact test was used to compare the compliance of reagents A and B with NAT reactivity, and the difference was not statistically significant (P<0.05). Among 14 HBsAg+ /NAT+ samples retested by CLIA, 2 were HBsAg reactive(14.29%, 2/14), 13 were anti-HBc reactive (92.86%, 13/14), 9 had the quantitative value of anti-HBs <10 mIU/mL, 5 had the quantitative value of anti-HBs between 10 to 100mIU/ mL. A total of 5 serological patterns were detected, and anti-HBe+ /anti-HBc+ pattern was the dominant. There were 262 cases of HBsAg+ /NAT- samples, but only 1 (0.38%, , 1/262) case was HBsAg reactive by CLIA, 100 were anti-HBc reactive (38.17%, 100/262), 144 (54.96%, 144/262) were anti-HBs reactive, and 1 was HBeAg reactive. A total of 8 serological patterns were detected. 【Conclusion】 Most of HBsAg single-ELISA-reactive results are false, and NAT could effectively reduce the residual risk of transfusion transmitted diseases.