Objective To explore the effect of heat inactivation on blood transfusion-related infection markers in serum and whole blood specimens. Methods A total of 57 positive serum specimens of hepatitis B virus surface antigen (HBsAg), anti-hepatitis C virus antibody (anti-HCV), anti-human immunodeficiency virus antibody (anti-HIV), anti-Treponema pallidum antibody (anti-TP), HBV DNA and HCV RNA as well as a total of 38 positive whole blood specimens of HBsAg, anti-HCV, anti-TP and HBV DNA were collected. After specimens were inactivated in a water bath at 56℃ for 30 minutes (which can effectively inactivate coronavirus), chemiluminescence and fluorescence quantitative polymerase chain reaction were adopted to detect the corresponding markers, differences among indicators before and after heat inactivation were compared. Results After serum was inactivated, detected results of HBsAg, anti-HCV, anti-HIV, anti-TP, HBV DNA and HCV RNA were not significantly different from those before inactivation (all P>0.05); after whole blood was inactivated, detection results of HBsAg, anti-HCV, anti-TP and HBV DNA were not statistically different from those before inactivation (all P>0.05); Bland-Altman scatterplot showed that all the difference data were within 95% limits of agreement (95% LoA), there was a good consistency in each indicator before and after inactivation. Conclusion The treatment method of heating at 56℃ water bath for 30 minutes has no obvious effect on the detection of blood transfusion-related infection markers, and can be widely used during the epidemic of coronavirus disease 2019.