To ensure safe blood transfusion, since June 2015, Nucleic Acid Testing (NAT) technique has been applied in blood screening process in some areas in Vietnam. The objective of our project is to examine the effectiveness of this technique, to provide specific, updated evidence, as the fundamental for promoting the application of NAT technology in screening blood donors. The study describes a series of cases, performed on 412,705 blood donated samples meeting inclusion criteria at Blood Transfusion Hematology Hospital from January 1, 2017 to January 1, 2019. All collected blood samples were screened for HBV, HCV, and HIV by serology technique, then samples with negative results will continue to be screened by NAT technique. Test results and donor information will be stored on the the hospital’s database. As a result, thanks to NAT screening, our study found 369 more positive blood samples with viral genetic material that serology did not detect, of which: 352 samples reacted with HBV-DNA (0.0871%), 10 samples responded to HCV-RNA (0.0025%) and 6 samples responded to HIV-RNA (0.0017%). Therefore, the application of NAT to the blood screening process is necessary to improve the safety of blood transfusion. Regarding the characteristics of positive cases with infectious agents, we recorded a higher rate of positive for HBV, HIV than men, even when tested by serology or by NAT technique (statistically significant, P <0.05); The positive rate for these 3 agents was higher for first-time blood donors than for repeat blood donors, statistically significant with serological screening (P <, 001) and not statistically significant when screening NAT (P> 0.05). In conclusion, The application of NAT technology in blood screening is essential and effective, improving blood transfusion safety.