Objective To know about the Babesia infection situation in the blood donors in Guangxi, China, so as to provide a basical epidemical date, and to provide scientific proofs for safety blood transfusion. Methods A total of 1 900 peripheral blood samples were obtained from tubes of used blood bagstaking from Guangxi, China in 2013. Babesia spp. infections in the donors were tested using Babesia 18S rRNA and β-tubulin Nest-PCR, morphological observation, and indirect immunofluorescence assay (IFA). Results We found that the positive rate of Babesia infection was 2.53% (48/1 900) in Guangxi, with all cases caused by Babesia microti. The PCR sensitivity of Babesia 18S rRNA primer was much higher than that of β-tubulin. In 38 blood the erythrocytes were found to have circular and dense nuclear, with ring-form thin cytoplasm under microscope. The specific fluorescence was not observed when Nest-PCR positive specimens with IFA at ≥1:64 antibody concentration, and was taken as negative. Conclusion There is a certain proportion of Babesia microti infection in Guangxi. For those patients with immunocompromised condition who needs blood transfusion, Babesia microti should been tested in blood donors.