Objective:Analysis of hepatitis B surface antigen negative donations, initial detection of reactivity followed by subsequent differential tests or repeat the infection status of non-discriminated reactive (NDR) unresponsive blood donors to explore whether different nucleic acid detection systems have differences in the detection of HBsAg -/NDR. Methods:The test result and reactivity rates of blood donor samples received from January 2020 to August 2022 were compared, and some samples were repeated for both nucleic acid testing systems, and the characteristics of the HBsAg -/HBV DNA + blood donors were analyzed. Results:Through the analysis of nucleic acid result from January 2020 to August 2022, repeated nucleic acid detection can improve the detection of HBsAg -/NDR blood donors, and the detection rate is offset in different cycle threshold valve ( Ct value) intervals or different sample absorbance to cut-off absorbance ratio ( S/ CO value) interval. Also grouping of Ct values or S/ CO values revealed that there was variability in detection rates between intervals in the two nucleic acid detection systems (PCR: X2=108.23, P<0.001, TMA: X2=40.95, P<0.001), mainly concentrated in Ct values less than 38.5 or S/ CO values between 10 and 15. And analysis of selected HBsAg -/HBV DNA + blood donor populations revealed that repeat nucleic acid testing was found for donors of different age ( X2=9.38, P=0.025), number of blood donations ( X2=22.52, P<0.001), occupation ( X2=24.92, P=0.002) and education level ( X2=10.37, P=0.016) significant differences, while there was no statistically significant difference for blood donors by gender ( X2=9.38, P>0.05), with far more males (67.50%) than females. Conclusions:For blood donors with a Ct value of less than 38.5, or an S/ CO value between 10 and 15, or a blood donor at the age of 41 to 50 years, another detection technology can be combined to improve the detection rate, reduce the potential risk of virus transmission of HBsAg -/NDR blood donors, and ensure the safety of clinical blood.