BackgroundMosquito-borne pathogens pose a significant threat to both human health and blood safety. The primary mosquito-borne viruses that present this threat are Zika virus, Chikungunya virus, and Dengue virus. At present, there are limited efficacious vaccines or therapeutic drugs for the prevention and treatment of these viral infections. Blood donors can remain asymptomatically infected and unfortunately, screening for these three viruses in Chinese blood donors are not mandatory, leaving the residual risk to transfusion recipients uncertain. Objective: To address this, we developed a single-tube multiplex RT-qPCR assay for ZCD detection and was preliminarily employed to screen a total of 10,566 blood donations in Nanning Blood Center in order to assess the prevalence risk of these pathogens in blood donors. Results: None of the blood samples was reactive for ZCD by nucleic acid test (NAT). One out of 173 donations (1/173, 0.58 %) was IgG positive for ZIKV and 14 (14/173, 8.4 %) were IgG positive for DENV. None of these 173 donations was IgG positive for Chikungunya virus. These findings suggest that the prevalence of ZCD infection in blood donors in Nanning is very low although past DENV infection (IgG positive) was relatively common. Conclusion: A single-tube multiplex RT-qPCR assay for simultaneous detection of ZCD viruses was successfully established and applied for screening in blood donors. The residual risk of ZCD infection through transfusion is currently low in Nanning, China. The NAT assay for ZCD will serve as a technical reserve in response to future epidemic or pandemic of mosquito-borne pathogens.