Objective To explore the optimization of blood testing strategies for voluntary blood donors in military blood centers. Methods From January 2017 to December 2020, the testing results of five serological indicators of voluntary blood donors from our center were collected via the blood transfusion management system . The positive NAT results of HBV, HCV and HIV, as well as the unqualified serological indicators, including the unqualified rate over the years, unqualified rate of military/civilian blood donor, reactive rate of dual-ELISA-reagent or one-ELISA-reagent etc., were retrospectively analyzed. Results The ratio of military blood donors to civilian blood donors in our center was 1: 4 during 2017~2020. The total unqualified rate of blood testing indicators of military blood donors was ALT>anti-HCV>HBsAg>HIV antigen/anti-HIV>anti-TP>NAT, and that of civilian blood donors was HBsAg > anti-TP > ALT > anti-HCV > HIV antigen/anti-HIV > NAT. The total unqualified rate of HBsAg, anti-HCV and anti-TP in our center decreased year by year, the HIV antigen/anti-HIV fluctuated, and the ALT increased year by year (P<0.01). The ALT deferral rate among military blood donors was the highest, accounting for 65.85%(594/902) of the donation deferral. The reactive rate of dual-ELISA-reagent to HBsAg, anti-HCV, HIV antigen/anti-HIV and anti-TP in civilian blood donors were 72.87%, 33.02%, 16.56% and 67.67%, respectively (P<0.05). Among the 123 668 ELISA negative samples during 2017~2020, 56 cases of HBV DNA(+ ) were detected by NAT, including 3 cases of military blood donors. Conclusion The current blood screening strategy of military blood centers can basically guarantee the safety of blood use in clinical. For military blood donors, ALT deferral, caused by ALT results that are higher than the ALT threshold, was the main reason for blood scrapping. Strengthening the promotion and implementation of NAT, as well as adjusting the critical value of ALT is of positive significance for reducing the risk of blood transfusion-transmitted diseases and the waste of blood sources, thereby improving the safety and security level of the clinical blood supply for the troops, promoting the treatment capabilities of officers and soldiers, and even improving the combat effectiveness of the troops.